Methods and compositions for treating asthma using anti-il-13 antibodies

ABSTRACT

The invention provides methods and compositions for treating asthma, e.g., mild or moderate asthma, in a subject using an anti-IL-13 antibody, or antigen-binding portion thereof.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/507,347, filed on Jul. 13, 2011, the entire contents of which arehereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Asthma is a chronic inflammatory disorder of the airways characterizedby wheezing, breathlessness, chest tightness, and cough. Asthma affectsapproximately 20 million people in the US, and about 75% of asthmapatients are adults. Of the adult asthma patients, approximately 60% ofasthma patients have mild disease, about 20% have moderate disease andthe remaining 20% have severe disease.

Interleukin-13 (IL-13) is thought to be pivotal in the pathogenesis ofhuman asthma, in that elevated levels of IL-13 are present in the lungsof asthma patients, and these levels correlate with disease severity(FIG. 1). Likewise, increased IL-13 is present in both sputum and inlung biopsies of patients with moderate to severe asthma who are treatedwith inhaled corticosteroids (ICS) or systemic corticosteroids andcontinue to be symptomatic. Moreover, human IL-13 genetic polymorphismsare associated with asthma and atopy (allergic hypersensitivity). IL-13binds to two receptors, IL-13Rα1 and IL-13Rα2. IL-13 is a well-validatedtarget for asthma as efficacy has been demonstrated using various meansof IL-13 antagonism in multiple, pre-clinical models of asthma.

Due to the role of human IL-13 in a variety of human disorders,therapeutic strategies have been designed to inhibit or counteract IL-13activity. In particular, antibodies that bind to, and neutralize, IL-13have been sought as a means to inhibit IL-13 activity. However, thereexists a need in the art for improved antibodies capable of bindingIL-13 for treating asthma.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treatingasthma, e.g., mild or moderate asthma, using an anti-IL-13 antibody, orantigen-binding portion thereof.

In one aspect, the invention provides an isolated composition comprisingan anti-IL-13 antibody, or antigen-binding portion thereof, wherein,when administered intravenously to a subject at a dose of about 0.3mg/kg, the antibody, or antigen-binding portion thereof, is capable ofexhibiting: (a) an area under the curve (AUC) of between about 1,500 andabout 2,700 μgh/ml; (b) a volume of distribution of between about 65 and125 mL/kg; (c) a peak concentration (C_(max)) of between about 5 andabout 8 μg/ml; and (d) a clearance rate of between about 0.1 and about0.2 ml/h/kg.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,wherein, when administered intravenously to a subject at a dose of about3 mg/kg, the antibody, or antigen-binding portion thereof, is capable ofexhibiting: (a) an area under the curve (AUC) of between about 21,000and about 33,500 μgh/ml; (b) a volume of distribution of between about55 and about 100 mL/kg; (c) a peak concentration (C_(max)) of betweenabout 55 and about 90 μg/ml; and (d) a clearance rate of between about0.08 and about 0.15 ml/h/kg.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,wherein, when administered intravenously to a subject at a dose of about10 mg/kg, the antibody, or antigen-binding portion thereof, is capableof exhibiting: (a) an area under the curve (AUC) of between about 75 andabout 100 μgh/ml; (b) a volume of distribution of between about 90 andabout 130 mL/kg; (c) a peak concentration (C_(max)) of between about 185and about 250 μg/ml; and (d) a clearance rate of between about 0.1 andabout 0.15 ml/h/kg.

In yet another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,wherein, when administered subcutaneously to a subject at a dose ofabout 0.3 mg/kg, the antibody, or antigen-binding portion thereof, iscapable of exhibiting: (a) an area under the curve (AUC) of betweenabout 125 and about 800 μgh/ml; and (b) a peak concentration (C_(max))of between about 1.0 and about 6.0 μg/ml.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,wherein, when administered subcutaneously to a subject at a dose ofabout 3 mg/kg, the antibody, or antigen-binding portion thereof, iscapable of exhibiting: (a) an area under the curve (AUC) of betweenabout 1,100 and about 8,500 μgh/ml; and (b) a peak concentration(C_(max)) of between about 12 and about 60 μg/ml.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, is 13C5.5, or an antigen-binding portion thereof. In anotherembodiment, the composition is a pharmaceutical composition.

In another aspect, the invention provides methods of treating orpreventing asthma in a subject by administering a composition of theinvention to the subject, thereby treating or preventing asthma. In oneembodiment, the composition is administered once. In another embodiment,the composition is administered weekly. In yet another embodiment, thecomposition is administered for about 3 weeks.

In one embodiment, the asthma is mild to moderate asthma. In anotherembodiment, the subject is a human.

In another embodiment, the method further comprises the administrationof an additional agent. In one embodiment, the additional agent isselected from the group consisting of: a therapeutic agent, an imagingagent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor,a co-stimulation molecule blocker, an adhesion molecule blocker, ananti-cytokine antibody or functional fragment thereof; methotrexate, acyclosporin, a rapamycin, an FK506, a detectable label or reporter, aTNF antagonist, an anti-rheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NTHE), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an oralsteroid, an epinephrine or analog, a cytokine, and a cytokineantagonist.

In another aspect, the invention provides methods of treating asthma ina subject by intravenously administering to the subject an anti-IL-13antibody, or antigen-binding portion thereof, wherein at least onepharmacokinetic characteristic selected from the group consisting of:(a) a maximum serum concentration (C_(max)) of between about 5 and about235 μg/mL, and (b) an area under the serum concentration-time curve(AUC) of between about 1,500 and about 98,000 μgh/mL, is achievedfollowing administration of the antibody, or antigen-binding portionthereof to the subject.

In one embodiment, the antibody, or antigen-binding portion thereof, isadministered at a dose of about 0.3 mg/kg. In one embodiment, theC_(max) is between about 5 and about 10 μg/mL. In one embodiment, theAUC is between about 1,500 and about 2,700 μgh/mL.

In another embodiment, the antibody, or antigen-binding portion thereof,is administered at a dose of about 3 mg/kg. In one embodiment, theC_(max) is between about 55 and about 90 μg/mL. In another embodiment,the AUC is between about 20,000 and about 34,000 μgh/mL.

In another embodiment, the antibody, or antigen-binding portion thereof,is administered at a dose of about 10 mg/kg. In one embodiment, theC_(max) is between about 190 and about 235 μg/mL. In one embodiment, theAUC is between about 75,000 and about 100,000 μgh/mL.

In another embodiment, the C_(max) value is between about 20 and about30 (μg/mL)/(mg/kg) after dose normalization. In another embodiment, theAUC is between about 6,000 and about 10,000 (μgh/mL)/(mg/kg) after dosenormalization.

In another aspect, the invention provides methods of treating asthma ina subject by subcutaneously administering to the subject an anti-IL-13antibody, or antigen-binding portion thereof, wherein at least onepharmacokinetic characteristic selected from the group consisting of:(a) a maximum serum concentration (C_(max)) of between about 1 and about60 μg/mL, and (b) an area under the serum concentration-time curve (AUC)of between about 125 and about 8,100 μgh/mL, is achieved following toadministration of the antibody, or antigen-binding portion thereof tothe subject.

In one embodiment, the antibody, or antigen-binding portion thereof, isadministered at a dose of about 0.3 mg/kg. In one embodiment, theC_(max) is between about 1 and about 6 μg/mL. In another embodiment, theAUC is between about 100 and about 800 μgh/mL.

In another embodiment, the antibody, or antigen-binding portion thereof,is administered at a dose of about 3 mg/kg. In one embodiment, theC_(max) is between about 12 and about 60 μg/mL. In another embodiment,the AUC is between about 1,100 and about 8,100 μgh/mL.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, is 13C5.5, or an antigen-binding portion thereof. In anotherembodiment, the subject is a human. In another embodiment, theanti-IL-13 antibody, or antigen-binding portion thereof, is administeredonce. In another embodiment, the anti-IL-13 antibody, or antigen-bindingportion thereof, is administered weekly. In yet another embodiment, theanti-IL-13 antibody, or antigen-binding portion thereof, is administeredfor three weeks.

In one embodiment, the asthma is mild to moderate asthma.

In another embodiment, the method further comprises the administrationof an additional agent. In one embodiment, the additional agent isselected from the group consisting of: a therapeutic agent, an imagingagent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor,a co-stimulation molecule blocker, an adhesion molecule blocker, ananti-cytokine antibody or functional fragment thereof; methotrexate, acyclosporin, a rapamycin, an FK506, a detectable label or reporter, aTNF antagonist, an anti-rheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NTHE), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an oralsteroid, an epinephrine or analog, a cytokine, and a cytokineantagonist.

In another aspect, the invention provides methods for treating asthma ina subject by subcutaneously administering to the subject an anti-IL-13antibody, or antigen-binding portion thereof, at a dose of about 0.3mg/kg, wherein at least one pharmacokinetic characteristic selected fromthe group consisting of: (a) a half-life of between about 24 and 31days; (b) a T_(max) of between about 3 and about 5 days; and (c) abioavailability of at least about 60% is achieved followingadministration of the antibody, or antigen-binding portion thereof tothe subject. In one embodiment, the bioavailability is at least about70%.

In another aspect, the invention provides methods of treating asthma ina subject by subcutaneously administering to the subject an anti-IL-13antibody, or antigen-binding portion thereof, at a dose of about 3mg/kg, wherein at least one pharmacokinetic characteristic selected fromthe group consisting of: (a) a half-life of between about 23 and 26days; (b) a T_(max) of less than or equal to about 5 days; and (c) abioavailability of at least about 60% is achieved followingadministration of the antibody, or antigen-binding portion thereof tothe subject. In one embodiment, the bioavailability is at least about70%.

In another aspect, the invention provides methods of treating asthma ina subject comprising intravenously administering to the subject ananti-IL-13 antibody, or antigen-binding portion thereof, at a dose ofabout 0.3 mg/kg, wherein at least one pharmacokinetic characteristicselected from the group consisting of: (a) a clearance rate of betweenabout 0.11 to about 0.19 mL/hr/kg; and (b) a volume of distribution ofbetween about 70 to about 130 mL/kg is achieved following administrationof the antibody, or antigen-binding portion thereof to the subject.

In another aspect, the invention provides methods treating asthma in asubject by intravenously administering to the subject an anti-IL-13antibody, or antigen-binding portion thereof, at a dose of about 3mg/kg, wherein at least one pharmacokinetic characteristic selected fromthe group consisting of: (a) a clearance rate of between about 0.08 toabout 0.14 mL/hr/kg; and (b) a volume of distribution of between about55 to about 100 mL/kg is achieved following administration of theantibody, or antigen-binding portion thereof to the subject.

In another aspect, the invention provides methods of treating asthma ina subject comprising intravenously administering to the subject ananti-IL-13 antibody, or antigen-binding portion thereof, at a dose ofabout 10 mg/kg, wherein at least one pharmacokinetic characteristicselected from the group consisting of: (a) a clearance rate of betweenabout 0.09 to about 0.13 mL/hr/kg; and (b) a volume of distribution ofbetween about 85 to about 130 mL/kg is achieved following administrationof the antibody, or antigen-binding portion thereof to the subject.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, is 13C5.5, or an antigen-binding portion thereof. In anotherembodiment, the subject is a human. In one embodiment, the anti-IL-13antibody, or antigen-binding portion thereof, is administered once. Inanother embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, is administered weekly. In yet another embodiment, theanti-IL-13 antibody, or antigen-binding portion thereof, is administeredfor 3 weeks.

In one embodiment, the asthma is mild to moderate asthma.

In another embodiment, the methods further comprise the administrationof an additional agent. In one embodiment, the additional agent isselected from the group consisting of: a therapeutic agent, an imagingagent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor,a co-stimulation molecule blocker, an adhesion molecule blocker, ananti-cytokine antibody or functional fragment thereof; methotrexate, acyclosporin, a rapamycin, an FK506, a detectable label or reporter, aTNF antagonist, an anti-rheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NTHE), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an oralsteroid, an epinephrine or analog, a cytokine, and a cytokineantagonist.

In one embodiment, the subject is a human.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,wherein, when administered intravenously to a subject at a dose of about0.3 mg/kg, 1 mg/kg, 3 mg/kg or 10 mg/kg, the antibody, orantigen-binding portion thereof, is capable of exhibiting any of thepharmacokinetic parameters set forth in the specification, Tables orFigures.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,wherein, when administered subcutaneously to a subject at a dose ofabout 0.3 mg/kg, 1 mg/kg or 3 mg/kg, the antibody, or antigen-bindingportion thereof, is capable of exhibiting any of the pharmacokineticparameters set forth in the specification, Tables or Figures.

In another aspect, the invention provides methods of treating orpreventing asthma in a subject by intravenously administering to thesubject an anti-IL-13 antibody, or antigen-binding portion thereof, at adose of about 0.3 mg/kg, 1 mg/kg, 3 mg/kg or 10 mg/kg, wherein at leastone of the pharmacokinetic characteristics set forth in thespecification, Tables or Figures is achieved following administration ofthe antibody, or antigen-binding portion thereof, to the subject.

In yet another aspect, the invention provides methods of treating orpreventing asthma in a subject by subcutaneously administering to thesubject an anti-IL-13 antibody, or antigen-binding portion thereof, at adose of about 0.3 mg/kg, 1 mg/kg, 3 mg/kg or 10 mg/kg, wherein at leastone of the pharmacokinetic characteristics set forth in thespecification, Tables or Figures is achieved following administration ofthe antibody, or antigen-binding portion thereof, to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting that IL-13 expression precedes pulmonarydysfunction.

FIG. 2 depicts that 13C5.5, an anti-IL-13 antibody, is derived from ahybridoma with a unique epitope and lineage.

FIG. 3 depicts that 13C5.5, an anti-IL-13 antibody, neutralizes IL-13 inthe lung.

FIG. 4 depicts the schematic of first in human (FIH) dosing used in thePhase I Clinical Trial.

FIG. 5 depicts the mean 13C5.5 serum concentration-time profile aftersingle 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg intravenous infusionsof 13C5.5 to healthy subjects on a linear scale.

FIG. 6 depicts the pharmacokinetic parameters of 13C5.5, an anti-IL-13antibody.

FIG. 7 depicts the mean 13C5.5 serum concentration-time profile aftersingle 0.3 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg intravenous infusions of13C5.5 to healthy and asthma subjects on a linear scale.

FIG. 8 depicts the pharmacokinetic parameters of 13C5.5, an anti-IL-13antibody.

FIG. 9 depicts the pharmacokinetic parameters of 13C5.5, an anti-IL-13antibody.

FIG. 10 depicts the pharmacokinetic parameters of 13C5.5, an anti-IL-13antibody. Bioavailability following subcutaneous administration wasestimated to be about 70%.

FIG. 11 depicts the mean dose normalized Cmax values following threeweekly 0.3 mg/kg (Group 8) and 3 mg/kg (Group 9) subcutaneous injectionsof 13C5.5 (Part 3 of the Clinical Trial).

FIG. 12 depicts the mean dose normalized AUC₀₋₁₆₈ values following threeweekly 0.3 mg/kg (Group 8) and 3 mg/kg (Group 9) subcutaneous injectionsof 13C5.5 (Part 3 of the Clinical Trial).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for treatingasthma, e.g., mild or moderate asthma, using an anti-IL-13 antibody, orantigen-binding portion thereof.

In order that the present invention may be more readily understood,certain terms are first defined.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

The terms “IL-13” and “IL-13 wild type” (abbreviated herein as IL-13,IL-13 wt), as used herein, include a cytokine that is secreted primarilyby T helper 2 cells. The term includes a monomeric protein of 13 kDapolypeptide. The structure of IL-13 is described further in, forexample, Moy, Diblasio et al. 2001 J Mol Biol 310 219-30. The term IL-13is intended to include recombinant human IL-13 (rh IL-13), which can beprepared by standard recombinant expression methods. The amino acidsequence of human IL-13, SEQ ID NO. 1, is known in the art.

Sequence of human IL-13 SEQ ID NO: 1MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

The term “IL-13 variant” (abbreviated herein as IL-13v), as used herein,includes a variant of IL-13 wherein amino acid residue 130 of SEQ IDNO:1 is changed from Arginine to Glutamine (R130Q).

“Biological activity” as used herein, refers to all inherent biologicalproperties of the cytokine. Biological properties of IL-13 include butare not limited to binding IL-13 receptor; (other examples includeimmunoglobulin isotype switching to IgE in human B cells and suppressinginflammatory cytokine production).

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Nonlimitingembodiments of which are discussed herein. In one embodiment, theantibody used in the compositions and methods of the invention is theanti-IL-13 antibody 13C5.5 described in U.S. Pat. No. 7,915,388,incorporated by reference herein. In another embodiment, the antibodyused in the compositions and methods of the invention is the antibody6A1, 3G4, tralokinumab, lebrikizumab, QAZ-576, IMA-638 or IMA-026.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 andIgA2) or subclass.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., IL-13). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.(ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Human IgG heavychain and light chain constant domain amino acid sequences are known inthe art and disclosed in Table 2 of U.S. Pat. No. 7,915,388, the entirecontents of which are incorporated herein by reference.

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds IL-13 is substantially free of antibodies that specifically bindantigens other than IL-13). An isolated antibody that specifically bindsIL-13 may, however, have cross-reactivity to other antigens, such asIL-13 molecules from other species. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in U.S. Pat. No. 7,915,388, the contents of which areincorporated herein by reference), antibodies isolated from arecombinant, combinatorial human antibody library (Hoogenboom H. R.,(1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin.Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002)BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) ImmunologyToday 21:371-378), antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., andGreen L. L. (2002) Current Opinion in Biotechnology 13:593-597; LittleM. et al (2000) Immunology Today 21:364-370) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo. One embodiment provides fully human antibodies capable of bindinghuman IL-13 which can be generated using techniques well known in theart, such as, but not limited to, using human Ig phage libraries such asthose disclosed in Jermutus et al., PCT publication No. WO 2005/007699A2.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences. In one embodiment, humanized anti human IL-13 antibodies andantigen binding portions are provided. Such antibodies were generated byobtaining murine anti-IL-13 monoclonal antibodies using traditionalhybridoma technology followed by humanization using in vitro geneticengineering, such as those disclosed in Kasaian et al PCT publicationNo. WO 2005/123126 A2.

The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% of the amino acid sequences ofone or more of the framework regions. In some embodiments, the term“acceptor” refers to the antibody amino acid or nucleic acid sequenceproviding or encoding the constant region(s). In yet another embodiment,the term “acceptor” refers to the antibody amino acid or nucleic acidsequence providing or encoding one or more of the framework regions andthe constant region(s). In a specific embodiment, the term “acceptor”refers to a human antibody amino acid or nucleic acid sequence thatprovides or encodes at least 80%, preferably, at least 85%, at least90%, at least 95%, at least 98%, or 100% of the amino acid sequences ofone or more of the framework regions. In accordance with thisembodiment, an acceptor may contain at least 1, at least 2, at least 3,least 4, at least 5, or at least 10 amino acid residues that does (do)not occur at one or more specific positions of a human antibody. Anacceptor framework region and/or acceptor constant region(s) may be,e.g., derived or obtained from a germline antibody gene, a matureantibody gene, a functional antibody (e.g., antibodies well-known in theart, antibodies in development, or antibodies commercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, although preferred embodiments useKabat or Chothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention the human heavy chain and lightchain acceptor sequences are selected from the sequences described inTable 3 and Table 4 disclosed in U.S. Pat. No. 7,915,388, the contentsof which are incorporated herein by reference.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med. Biol. 484:13-30(2001)). One of the advantages provided by various embodiments of thepresent invention stems from the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or fragment thereof which immunospecificallybinds to an antigen of interest and which comprises a framework (FR)region having substantially the amino acid sequence of a human antibodyand a complementary determining region (CDR) having substantially theamino acid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, preferably at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to the amino acid sequence of a non-human antibody CDR. Ahumanized antibody comprises substantially all of at least one, andtypically two, variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) in whichall or substantially all of the CDR regions correspond to those of anon-human immunoglobulin (i.e., donor antibody) and all or substantiallyall of the framework regions are those of a human immunoglobulinconsensus sequence. Preferably, a humanized antibody also comprises atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. In some embodiments, a humanizedantibody contains both the light chain as well as at least the variabledomain of a heavy chain. The antibody also may include the CH1, hinge,CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, ahumanized antibody only contains a humanized light chain. In someembodiments, a humanized antibody only contains a humanized heavy chain.In specific embodiments, a humanized antibody only contains a humanizedvariable domain of a light chain and/or humanized heavy chain.

In one embodiment of the invention, the humanized anti-IL-13 antibody is13C5.5. 13C5.5 has the sequences SEQ ID NO:2 (heavy chain variableregion) and SEQ ID NO:3 (light chain variable region). See also U.S.Pat. No. 7,915,388, the entire contents of which are incorporated hereinby reference.

Heavy Chain Variable Region 13C5.5 SEQ ID NO: 2EVTLRESGPGLVKPTQTLTLTCTLYGFSLSTSDMGVDWIRQPPGKGLEWLAHIWWDDVKRYNPALKSRLTISKDTSKNQVVLKLTSVDPVDTATYYCARTVSSGYIYYAMDYWGQGTLVTVSS Light Chain Variable Region 13C5.5 SEQ ID NO: 3DIQMTQSPSSLSASVGDRVTISCRASQDIRNYLNWYQQKPGKAPKLLIFYTSKLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQGNTLPLTFGG GTKVEIK

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “multivalent binding protein” is used in this specification todenote a binding protein comprising two or more antigen binding sites.The multivalent binding protein is preferably engineered to have thethree or more antigen binding sites, and is generally not a naturallyoccurring antibody. The term “multispecific binding protein” refers to abinding protein capable of binding two or more related or unrelatedtargets. Dual variable domain (DVD) binding proteins as used herein, arebinding proteins that comprise two or more antigen binding sites and aretetravalent or multivalent binding proteins. Such DVDs may bemonospecific, i.e. capable of binding one antigen or multispecific, i.e.capable of binding two or more antigens. DVD binding proteins comprisingtwo heavy chain DVD polypeptides and two light chain DVD polypeptidesare referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chainDVD polypeptide, and a light chain DVD polypeptide, and two antigenbinding sites. Each binding site comprises a heavy chain variable domainand a light chain variable domain with a total of 6 CDRs involved inantigen binding per antigen binding site.

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of a cytokine when a binding protein specificallybinds the cytokine. Preferably a neutralizing binding protein is aneutralizing antibody whose binding to IL-13 and/or IL-13 results ininhibition of a biological activity of IL-13 and/or IL-13. Preferablythe neutralizing binding protein binds IL-13 and/or IL-13 and reduces abiologically activity of IL-13 and/or IL-13 by at least about 20%, 40%,60%, 80%, 85% or more Inhibition of a biological activity of IL-13and/or IL-13 by a neutralizing binding protein can be assessed bymeasuring one or more indicators of IL-13 and/or IL-13 biologicalactivity well known in the art. For example, inhibition of human IL-13induced production of TARC(CCL-17) by A-549 cells can be measured (seeExample 1.1.0 of U.S. Pat. No. 7,915,388, the contents of which areincorporated herein by reference).

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-IL-13 antibody that binds to an IL-13 antigen and/or theneutralizing potency of an antibody, for example, an anti-IL-13 antibodywhose binding to IL-13 inhibits the biological activity of IL-13, e.g.For example inhibition of human IL-13 induced production of TARC(CCL-17)by A-549 cells (see Example 1.1.0 of U.S. Pat. No. 7,915,388, the entirecontents of which are incorporated herein by reference).

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann Biol. Clin.51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “k_(on),”, as used herein, is intended to refer to the on rateconstant for association of an antibody to the antigen to form theantibody/antigen complex as is known in the art.

The term “k_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex as is known in the art.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The term “labeled binding protein” as used herein, refers to a proteinwith a label incorporated that provides for the identification of thebinding protein. Preferably, the label is a detectable marker, e.g.,incorporation of a radiolabeled amino acid or attachment to apolypeptide of biotinyl moieties that can be detected by marked avidin(e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or colorimetric methods).Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y,99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm); fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof.

The terms “crystal”, and “crystallized” as used herein, refer to anantibody, or antigen binding portion thereof, that exists in the form ofa crystal. Crystals are one form of the solid state of matter, which isdistinct from other forms such as the amorphous solid state or theliquid crystalline state. Crystals are composed of regular, repeating,three-dimensional arrays of atoms, ions, molecules (e.g., proteins suchas antibodies), or molecular assemblies (e.g., antigen/antibodycomplexes). These three-dimensional arrays are arranged according tospecific mathematical relationships that are well-understood in thefield. The fundamental unit, or building block, that is repeated in acrystal is called the asymmetric unit. Repetition of the asymmetric unitin an arrangement that conforms to a given, well-definedcrystallographic symmetry provides the “unit cell” of the crystal.Repetition of the unit cell by regular translations in all threedimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett,Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2ndea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).”

The term “polynucleotide” as referred to herein means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide”: is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Protein constructs of the presentinvention may be expressed, and purified using expression vectors andhost cells known in the art, including expression cassettes, vectors,recombinant host cells and methods for the recombinant expression andproteolytic processing of recombinant polyproteins and pre-proteins froma single open reading frame (e.g., WO 2007/014162, the entire contentsof which are incorporated herein by reference).

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the Kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct, which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of IL-13).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of IL-13). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies are described, e.g., in WO01/83525.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist. Particular agonists of interest may include, but are notlimited to, IL-13 polypeptides or polypeptides, nucleic acids,carbohydrates, or any other molecules that bind to IL-13.

The term “antagonist” or “inhibitor”, as used herein, refer to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological or immunologicalactivity of IL-13 and/or IL-13. Antagonists and inhibitors of IL-13and/or IL-13 may include, but are not limited to, proteins; nucleicacids, carbohydrates, or any other molecules, which bind to IL-13 and/orIL-13.

The term “inhibit binding to the receptor” refers to the ability of thebinding protein to prevent the binding of IL-13 to one or more of itsreceptors. Such inhibition of binding to the receptor would result indiminishing or abolishing the biological activity mediated by binding ofIL-13 to its receptor or receptors.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, serum, urine, synovial fluid, cells,organs, tissues, bone marrow, lymph nodes and spleen.

The term “C_(max)” refers to the maximum or peak serum or plasmaconcentration of an agent observed in a subject after itsadministration.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, of the invention (e.g., a humanized anti-IL-13 antibody such as13C5.5, or an antigen-binding portion thereof) is administeredintravenously and exhibits a maximum serum concentration (C_(max)) ofbetween about 5 and about 235 μg/mL; a peak concentration (C_(max)) ofbetween about 5 and about 8 μg/ml; a C_(max) of between about 5 andabout 10 μg/mL; a peak concentration (C_(max)) of between about 55 andabout 90 μg/ml; a peak concentration (C_(max)) of between about 185 andabout 250 μg/ml; a C_(max) of between about 190 and about 235 μg/mL. Inanother embodiment, the C_(max) is between about 5 and about 50, betweenabout 50 and about 75, between about 75 and about 100, between about 100and about 125, between about 125 and about 150, between about 150 andabout 175, between about 175 and about 200, or between about 200 andabout 235 μg/mL.

In another embodiment, the anti-IL-13 antibody, or antigen-bindingportion thereof (e.g., a humanized anti-IL-13 antibody such as 13C5.5,or an antigen-binding portion thereof), is administered intravenouslyand exhibits a C_(max) value of between about 20 and about 30(μg/mL)/(mg/kg) after dose normalization. In another embodiment, theanti-IL-13 antibody, or antigen-binding portion thereof, is administeredintravenously and exhibits a C_(max) value of about 20, about 21, about22, about 23, about 24, about 25, about 26, about 27, about 28, about 29and about 30 (μg/mL)/(mg/kg) after dose normalization. In anotherembodiment, the anti-IL-13 antibody, or antigen-binding portion thereof,is administered intravenously and exhibits a C_(max) value of betweenabout 10 and about 40 (μg/mL)/(mg/kg) after dose normalization.

In another embodiment, the anti-IL-13 antibody, or antigen-bindingportion thereof, of the invention (e.g., a humanized anti-IL-13 antibodysuch as 13C5.5, or an antigen-binding portion thereof) is administeredsubcutaneously and exhibits a maximum serum concentration (C_(max)) ofbetween about 1 and about 60 μg/mL; a peak concentration (C_(max)) ofbetween about 1.0 and about 6.0 μg/ml; a C_(max) value of between about6 and about 12 μg/ml; a peak concentration (C_(max)) of between about 12and about 60 μg/ml; a C_(max) value of between about 1 and about 10,between about 10 and about 20, between about 20 and about 30, betweenabout 30 and about 40, between about 40 and about 50, between about 50and about 60, between about 20 and about 60, or between about 40 andabout 60 μg/ml.

The term “T_(max)” refers to the time at which C_(max) occurred. In oneembodiment, the anti-IL-13 antibody, or antigen-binding portion thereof,of the invention (e.g., a humanized anti-IL-13 antibody such as 13C5.5,or an antigen-binding portion thereof) is administered intravenously orsubcutaneously and exhibits a T_(max) of between about 1 and about 5days; a T_(max) of between about 3 and about 5 days; a T_(max) of lessthan or equal to about 5 days; a T_(max) of about 1 day, a T_(max) ofabout 2 days, a T_(max) of about 3 days, a T_(max) of about 4 days, aT_(max) of about 5 days, a T_(max) of about 6 days, a T_(max) of about 7days, a T_(max) of about 8 days, a T_(max) of about 9 days, or a T_(max)of about 10 days.

The term “bioavailability” or “F %” refers to a fraction or percent of adose which is absorbed and enters the systemic circulation afteradministration of a given dosage form. The dose of the anti-IL-13antibody, or antigen-binding portion thereof, may be administeredthrough any route, and, preferably, via intravenous or subcutaneousinjection. In one embodiment, the anti-IL-13 antibody, orantigen-binding portion thereof, of the invention (e.g., a humanizedanti-IL-13 antibody such as 13C5.5, or an antigen-binding portionthereof) is administered intravenously or subcutaneously and exhibits abioavailability of at least about 60%. In another embodiment, theantibody, or antigen-binding portion thereof, exhibits a bioavailabilityof at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, or at least about 100%.

The term “AUC” or “area under the curve” is related to clearance. Ahigher clearance rate is related to a smaller AUC, and a lower clearancerate is related to a larger AUC value. The AUC higher values representslower clearance rates.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, of the invention (e.g., a humanized anti-IL-13 antibody such as13C5.5, or an antigen-binding portion thereof) is administeredintravenously and exhibits an area under the curve (AUC) of betweenabout 75 and about 100,000 μgh/mL; an AUC of between about 75 and about100; between about 1,500 and about 2,700 μgh/ml; between about 1,500 andabout 3,000; between about 21,000 and about 33,500 μgh/ml; between about1,500 and 98,000; between about 20,000 and about 34,000 μgh/mL; betweenabout 34,000 and about 40,000; between about 40,000 and about 50,000;between about 50,000 and about 60,000; between about 60,000 and about75,000; between about 75,000 and about 100,000 μgh/mL; about 75, about100, about 150, about 200, about 250, about 300, about 350, about 400,about 450, about 500, about 550, about 600, about 650, about 700, about750, about 800, about 850, about 900, about 950, about 1,000; about1,100; about 1,200; about 1,300; about 1,400; about 1,500; about 1,600;about 1,700; about 1,800; about 1,900; about 2,000; about 2,250; about2,500; about 2,750; about 3,000; about 4,000; about 5,000; about 6,000;about 7,000; about 8,000; about 9,000; about 10,000; about 12,000; about15,000; about 20,000; about 25,000; about 30,000; about 35,000; about40,000; about 45,000; about 50,000; about 55,000; about 60,000; about65,000; about 70,000; about 75,000; about 80,000, about 85,000; about90,000; about 95,000 or about 100,000 μgh/mL.

In another embodiment, the AUC is between about 6,000 and about 10,000(μgh/mL)/(mg/kg) after dose normalization, between about 7,000 and about9,000; about 6,000; about 6,500; about 7,000; about 7,500; about 8,000;about 8,500; about 9,000; about 9,500 or about 10,000 (μgh/mL)/(mg/kg)after dose normalization.

In another embodiment, the anti-IL-13 antibody, or antigen-bindingportion thereof, of the invention (e.g., a humanized anti-IL-13 antibodysuch as 13C5.5, or an antigen-binding portion thereof) is administeredsubcutaneously and exhibits an area under the curve (AUC) of betweenabout 125 and about 8,100 μgh/mL; between about 125 and about 800μgh/ml; between about 800 and about 1,100; between about 1,100 and about8,100; about 100 and about 800 μgh/mL; about 125; about 150; about 175;about 200; about 250; about 300; about 350; about 400; about 450; about500; about 550; about 600; about 650; about 700; about 750; about 800;about 850; about 900; about 950; about 1,000; about 1,100; about 1,200;about 1,300; about 1,400; about 1,500; about 1,600; about 1,700; about1,800; about 1,900; about 2,000; about 2,250; about 2,500; about 3,000;about 3,500; about 4,000; about 4,500; about 5,000; about 5,500; about6,000; about 6,500; about 7,000; about 7,500; about 8,000 or about 8,100μgh/mL.

As used herein, the term “clearance rate” is related to the AUC, or areaunder the curve. A higher clearance rate is related to a smaller AUC,and a lower clearance rate is related to a larger AUC value. The AUChigher values represent slower clearance rates.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, of the invention (e.g., a humanized anti-IL-13 antibody such as13C5.5, or an antigen-binding portion thereof) is administeredintravenously and exhibits a clearance rate of between about 0.08 andabout 0.2 ml/h/kg, between about 0.08 and about 0.15 ml/h/kg; betweenabout 0.1 and about 0.15 ml/h/kg; between about 0.11 to about 0.19mL/hr/kg; between about 0.08 to about 0.14 mL/hr/kg; between about 0.09to about 0.13 mL/hr/kg; about 0.08; about 0.09, about 0.1, about 0.11,about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17,about 0.18, about 0.19, about 2.0, about 2.1, about 2.2, about 2.3,about 2.4 or about 2.5 mL/h/kg.

As used herein, the term “volume of distribution” is a term used toquantify the distribution of a drug, e.g., an anti-IL-13 antibody, orantigen-binding portion thereof, between plasma and the rest of the bodyafter dosing. The volume of distribution is the theoretical volume inwhich the total amount of drug would need to be uniformly distributed inorder to produced the desired blood concentration of the drug.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, of the invention (e.g., a humanized anti-IL-13 antibody such as13C5.5, or an antigen-binding portion thereof) is administeredintravenously and exhibits a volume of distribution of between about 55and about 130 mL/kg; between about 65 and 125 mL/kg; between about 55and about 100 mL/kg; between about 90 and about 130 mL/kg; between about70 to about 130 mL/kg; between about 85 to about 130 mL/kg; betweenabout 100 to about 130; between about 110 to about 120; about 55, about60, about 65, about 70, about 75, about 80, about 85, about 90, about95, about 100, about 105, about 110, about 115, about 120, about 125,about 130 or about 135 mL/kg.

In one embodiment, the anti-IL-13 antibody, or antigen-binding portionthereof, of the invention (e.g., a humanized anti-IL-13 antibody such as13C5.5, or an antigen-binding portion thereof) is administeredintravenously or subcutaneously and has a half-life of between about 24and 31 days; between about 23 and 26 days; between about 10 and about 40days, between about 20 and about 30 days, about 10 days, about 15 days,about 16 days, about 17 days, about 18 days, about 19 days, about 20days, about 21 days, about 22 days, about 23 days, about 24 days, about25 days, about 26 days, about 27 days, about 28 days, about 29 days,about 30 days, about 31 days, about 32 days, about 33 days, about 34days, about 35 days, about 36 days, about 37 days, about 38 days, about39 days, or about 40 days.

The term “dosing” or “dose” or “dosage”, as used herein, refers to theadministration of a substance (e.g., an anti-IL-13 antibody, orantigen-binding portion thereof) to achieve a therapeutic objective(e.g., the treatment of asthma).

In one embodiment, the composition of the invention is administeredonce. In another embodiment, the composition of the invention isadministered weekly. In another embodiment, the composition of theinvention is administered for two weeks. In another embodiment, thecomposition of the invention is administered for three weeks. In anotherembodiment, the composition is administered for four weeks, five weeks,six weeks, seven weeks, eight weeks, nine weeks, ten weeks, elevenweeks, three months, four months, five months, six months, seven months,eight months, nine months, ten months, eleven months, twelve months,thirteen months, fourteen months, fifteen months, sixteen months,seventeen months, eighteen months, nineteen months, twenty months,twenty-one months, twenty-two months, twenty-three months, two years,three years, four years, five years, ten years, or for the life of thesubject.

The term “combination” as in the phrase “a first agent in combinationwith a second agent” includes co-administration of a first agent and asecond agent, which for example may be dissolved or intermixed in thesame pharmaceutically acceptable carrier, or administration of a firstagent, followed by the second agent, or administration of the secondagent, followed by the first agent. The present invention, therefore,includes methods of combination therapeutic treatment and combinationpharmaceutical compositions.

The term “concomitant” as in the phrase “concomitant therapeutictreatment” includes administering an agent in the presence of a secondagent. A concomitant therapeutic treatment method includes methods inwhich the first, second, third, or additional agents areco-administered. A concomitant therapeutic treatment method alsoincludes methods in which the first or additional agents areadministered in the presence of a second or additional agents, whereinthe second or additional agents, for example, may have been previouslyadministered. A concomitant therapeutic treatment method may be executedstep-wise by different actors. For example, one actor may administer toa subject a first agent and a second actor may to administer to thesubject a second agent, and the administering steps may be executed atthe same time, or nearly the same time, or at distant times, so long asthe first agent (and additional agents) are after administration in thepresence of the second agent (and additional agents). The actor and thesubject may be the same entity (e.g., human).

The term “combination therapy”, as used herein, refers to theadministration of two or more therapeutic substances, e.g., ananti-IL-13 antibody and another drug. The other drug(s) may beadministered concomitant with, prior to, or following the administrationof the anti-IL-13 antibody.

The term “kit” as used herein refers to a packaged product comprisingcomponents with which to administer the anti-IL-13 antibody of theinvention for treatment of a IL-13 related disorder. The kit preferablycomprises a box or container that holds the components of the kit. Thebox or container is affixed with a label or a Food and DrugAdministration approved protocol. The box or container holds componentsof the invention which are preferably contained within plastic,polyethylene, polypropylene, ethylene, or propylene vessels. The vesselscan be capped-tubes or bottles. The kit can also include instructionsfor administering an anti-IL-13 antibody.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Antibodies that Bind IL-13

This invention provides methods and compositions for using anti-IL-13antibodies, or antigen-binding portions thereof, for the treatment ofasthma. In one aspect, the present invention provides compositions whichinclude and/or methods which use isolated murine monoclonal antibodies,or antigen-binding portions thereof, that bind to IL-13 with highaffinity, a slow off rate and high neutralizing capacity. In a secondaspect, the invention provides compositions which include and/or methodswhich use chimeric antibodies that bind IL-13. In a third aspect, theinvention provides compositions which include and/or methods which usehumanized antibodies, or antigen-binding portions thereof, that bindIL-13. Preferably, the antibodies, or portions thereof, are isolatedantibodies. Preferably, the antibodies are neutralizing anti-IL-13and/or humanized or human anti-IL-13 antibodies.

A. Methods of Making Anti-IL-13 Antibodies

Antibodies to be used in the compositions and/or methods of the presentinvention may be made by any of a number of techniques known in the art.For example, they can be made using the techniques disclosed in U.S.Pat. No. 7,915,338, the entire contents of which are incorporated hereinby reference.

1. Anti-IL-13 Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention (See Example 1.2). Briefly, mice canbe immunized with an IL-13 antigen. In a preferred embodiment, the IL-13antigen is administered with an adjuvant to stimulate the immuneresponse. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks.

After immunization of an animal with an IL-13 antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-IL-13antibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-IL-13 antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen IL-13 are detected in the mouse serum, the mouse spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the ATCC. Hybridomas are selected andcloned by limited dilution. The hybridoma clones are then assayed bymethods known in the art for cells that secrete antibodies capable ofbinding IL-13. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using IL-13, or a portionthereof, or a cell expressing IL-13. In a preferred embodiment, theinitial screening is performed using an enzyme-linked immunoassay(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example ofELISA screening is provided in WO 00/37504, herein incorporated byreference.

Anti-IL-13 antibody-producing hybridomas are selected, cloned andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

In a preferred embodiment, the hybridomas are mouse hybridomas, asdescribed above. In another preferred embodiment, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-IL-13 antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

2. Anti-IL-13 Monoclonal Antibodies Using SLAM

Recombinant antibodies for use in the compositions and/or methods of thepresent invention may also be generated from single, isolatedlymphocytes using a procedure referred to in the art as the selectedlymphocyte antibody method (SLAM), as described in U.S. Pat. Nos.7,915,388 and 5,627,052, PCT Publication WO 92/02551 and Babcock, J. S.et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848, the entirecontents of each of which are incorporated herein by reference. Briefly,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals described above, are screenedusing an antigen-specific hemolytic plaque assay, wherein the antigenIL-13, a subunit of IL-13, or a fragment thereof, is coupled to sheepred blood cells using a linker, such as biotin, and used to identifysingle cells that secrete antibodies with specificity for IL-13.Following identification of antibody-secreting cells of interest, heavy-and light-chain variable region cDNAs are rescued from the cells byreverse transcriptase-PCR and these variable regions can then beexpressed, in the context of appropriate immunoglobulin constant regions(e.g., human constant regions), in mammalian host cells, such as COS orCHO cells. The host cells transfected with the amplified immunoglobulinsequences, derived from in vivo selected lymphocytes, can then undergofurther analysis and selection in vitro, for example by panning thetransfected cells to isolate cells expressing antibodies to IL-13. Theamplified immunoglobulin sequences further can be manipulated in vitro,such as by in vitro affinity maturation methods such as those describedin PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

3. Anti-IL-13 Monoclonal Antibodies Using Transgenic Animals

Antibodies for use in the compositions and/or methods of the presentinvention may also be produced by immunizing a non-human animalcomprising some, or all, of the human immunoglobulin locus with an IL-13antigen. In a preferred embodiment, the non-human animal is a XENOMOUSEtransgenic mouse, an engineered mouse strain that comprises largefragments of the human immunoglobulin loci and is deficient in mouseantibody production. See, e.g., Green et al. Nature Genetics 7:13-21(1994) and U.S. Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209,6,075,181, 6,091,001, 6,114,598 and 6,130,364. See also WO 91/10741,published Jul. 25, 1991, WO 94/02602, published Feb. 3, 1994, WO96/34096 and WO 96/33735, both published Oct. 31, 1996, WO 98/16654,published Apr. 23, 1998, WO 98/24893, published Jun. 11, 1998, WO98/50433, published Nov. 12, 1998, WO 99/45031, published Sep. 10, 1999,WO 99/53049, published Oct. 21, 1999, WO 00 09560, published Feb. 24,2000 and WO 00/037504, published Jun. 29, 2000, the entire contents ofeach of which are expressly incorporated herein by reference. TheXENOMOUSE transgenic mouse produces an adult-like human repertoire offully human antibodies, and generates antigen-specific human Mabs. TheXENOMOUSE transgenic mouse contains approximately 80% of the humanantibody repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics 15:146-156 (1997), Greenand Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of whichare hereby incorporated by reference.

4. Anti-IL-13 Monoclonal Antibodies Using Recombinant Antibody Libraries

In vitro methods also can be used to make the antibodies for use in thecompositions and/or methods of the invention, wherein an antibodylibrary is screened to identify an antibody having the desired bindingspecificity. Methods for such screening of recombinant antibodylibraries are well known in the art and include methods described in,for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTPublication No. WO 92/18619; Dower et al. PCT Publication No. WO91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al.PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard etal. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; McCafferty et al., Nature (1990)348:552-554; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, US patentapplication publication 20030186374, and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

The recombinant antibody library may be from a subject immunized withIL-13 or IL-13, or a portion of IL-13 or IL-13. Alternatively, therecombinant antibody library may be from a naive subject, i.e., one whohas not been immunized with IL-13, such as a human antibody library froma human subject who has not been immunized with human IL-13. Antibodiesof the invention are selected by screening the recombinant antibodylibrary with the peptide comprising human IL-13 to thereby select thoseantibodies that recognize IL-13. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for IL-13, such as thosethat dissociate from human IL-13 with a particular k.sub.off rateconstant, the art-known method of surface plasmon resonance can be usedto select antibodies having the desired k.sub.off rate constant. Toselect antibodies of the invention having a particular neutralizingactivity for IL-13, such as those with a particular an IC.sub.50,standard methods known in the art for assessing the inhibition of IL-13activity may be used.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human IL-13. Preferably, theantibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of dualspecificity antibodies of the invention. One type of alternativeexpression system is one in which the recombinant antibody library isexpressed as RNA-protein fusions, as described in PCT Publication No. WO98/31700 by Szostak and Roberts, and in Roberts, R. W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, acovalent fusion is created between an mRNA and the peptide or proteinthat it encodes by in vitro translation of synthetic mRNAs that carrypuromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, aspecific mRNA can be enriched from a complex mixture of mRNAs (e.g., acombinatorial library) based on the properties of the encoded peptide orprotein, e.g., antibody, or portion thereof, such as binding of theantibody, or portion thereof, to the dual specificity antigen. Nucleicacid sequences encoding antibodies, or portions thereof, recovered fromscreening of such libraries can be expressed by recombinant means asdescribed above (e.g., in mammalian host cells) and, moreover, can besubjected to further affinity maturation by either additional rounds ofscreening of mRNA-peptide fusions in which mutations have beenintroduced into the originally selected sequence(s), or by other methodsfor affinity maturation in vitro of recombinant antibodies, as describedabove.

In another approach the antibodies for use in the compositions and/ormethods of the present invention can also be generated using yeastdisplay methods known in the art. In yeast display methods, geneticmethods are used to tether antibody domains to the yeast cell wall anddisplay them on the surface of yeast. In particular, such yeast can beutilized to display antigen-binding domains expressed from a repertoireor combinatorial antibody library (e.g., human or murine). Examples ofyeast display methods that can be used to make the antibodies of thepresent invention include those disclosed Wittrup, et al. U.S. Pat. No.6,699,658 incorporated herein by reference.

B. Production of Recombinant IL-13 Antibodies

Antibodies for use in the compositions and/or methods of the presentinvention may be produced by any of a number of techniques known in theart. For example, expression from host cells, wherein expressionvector(s) encoding the heavy and light chains is (are) transfected intoa host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it ispossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and ChasM, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian to host cells, the antibodies are produced by culturingthe host cells for a period of time sufficient to allow for expressionof the antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

1. Anti IL-13 Antibodies

Table 5 of U.S. Pat. No. 7,195,388 (the contents of which areincorporated herein by reference) is a list of amino acid sequences ofVH and VL regions of preferred anti-IL-13 antibodies to be used in thecompositions and/or methods of the invention. These isolated anti-IL-13antibody CDR sequences establish a family of IL-13 binding proteins,isolated in accordance with this invention, and comprising polypeptidesthat include the CDR sequences listed in Table 6 of U.S. Pat. No.7,195,388 (the contents of which are incorporated herein by reference).To generate and to select CDRs of the invention having preferred IL-13binding and/or neutralizing activity with respect to IL-13 and/or IL-13,standard methods known in the art for generating binding proteins of thepresent invention and assessing the IL-13 and or IL-13 binding and/orneutralizing characteristics of those binding protein may be used,including but not limited to those specifically described herein.

In one embodiment, the antibody used in the compositions and/or methodsof the invention is the antibody 13C5.5 (see U.S. Pat. No. 7,915,388,the entire contents of which are incorporated herein by reference).13C5.5 is a humanized antibody that binds with great affinity to helicesA and D of interleukin 13 (IL-13) (see FIG. 1). The heavy and lightchain variable region sequences of 13C5.5 are set forth above as SEQ IDNO:2 and SEQ ID NO:3, respectively.

2. Anti IL-13 Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies are known in the art and discussed in to detail in Example2.1. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, whichare incorporated herein by reference in their entireties. In addition,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger etal., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454which are incorporated herein by reference in their entireties) bysplicing genes from a mouse antibody molecule of appropriate antigenspecificity together with genes from a human antibody molecule ofappropriate biological activity can be used.

In one embodiment, the chimeric antibodies for use in the compositionsand/or methods of the invention are produced by replacing the heavychain constant region of the murine monoclonal anti human IL-13antibodies described in section 1 with a human IgG1 constant region. Ina specific embodiment the chimeric antibody of the invention comprises aheavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO:46 and a light chain variable region (VL) comprising the amino acidsequence of SEQ ID NO: 35; SEQ ID NO: 37; SEQ ID NO: 40; SEQ ID NO: 43;or SEQ ID NO: 47 disclosed in U.S. Pat. No. 7,195,388.

3. Anti IL-13 Humanized Antibodies

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Known humanIg sequences are disclosed, e.g., Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Dept. Health (1983), entirely incorporatedherein by reference. Such imported sequences can be used to reduceimmunogenicity or reduce, enhance or modify binding, affinity, on-rate,off-rate, avidity, specificity, half-life, or any other suitablecharacteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994), Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

C. Production of Antibodies and Antibody-producing Cell Lines

Preferably, anti-IL-13 antibodies for use in the compositions and/ormethods of the present invention exhibit a high capacity to reduce or toneutralize IL-13 activity, e.g., as assessed by any one of several invitro and in vivo assays known in the art (e.g., see Example 1.1.0 ofU.S. Pat. No. 7,195,388, the entire contents of which are incorporatedherein by reference.). For example, these antibodies neutralizeIL-13-induced production of TARC by A-549 cells with IC₅₀ values in therange of at least about 10⁻⁸ M, about 10⁻⁹ M, or about 10⁻¹⁰ M.

In preferred embodiments, the isolated antibody, or antigen-bindingportion thereof, binds human IL-13, wherein the antibody, orantigen-binding portion thereof, dissociates from human IL-13 with ak_(off) rate constant of about 0.1 s⁻¹ or less, as determined by surfaceplasmon resonance, or which inhibits human IL-13 and/or human IL-13activity with an IC₅₀ of about 1×10.sup.-6M or less. Alternatively, theantibody, or an antigen-binding portion thereof, may dissociate fromhuman IL-13 with a k_(off) rate constant of about 1×10⁻² s⁻¹ or less, asdetermined by surface plasmon resonance, or may inhibit human IL-13and/or human IL-13 activity with an IC₅₀ of about 1×10⁻⁷M or less.Alternatively, the antibody, or an antigen-binding portion thereof, maydissociate from human IL-13 with a k_(off) rate constant of about 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance, or may inhibithuman IL-13 and/or human IL-13 with an IC₅₀ of about 1×10⁻⁸M or less.Alternatively, the antibody, or an antigen-binding portion thereof, maydissociate from human IL-13 with a k_(off) rate constant of about 1×10⁻⁴s⁻¹ or less, as determined by surface plasmon resonance, or may inhibitIL-13 and/or human L-13 activity with an IC₅₀ of about 1×10⁻⁹M or less.Alternatively, the antibody, or an antigen-binding portion thereof, maydissociate from human IL-13 with a k_(off) rate constant of about 1×10⁻⁵s⁻¹ or less, as determined by surface plasmon resonance, or may inhibitIL-13 and/or human IL-13 activity with an IC₅₀ of about 1×10⁻¹⁰M orless. Alternatively, the antibody, or an antigen-binding portionthereof, may dissociate from human IL-13 with a k_(off) rate constant ofabout 1×10⁻⁵ s⁻¹ or less, as determined by surface plasmon resonance, ormay inhibit IL-13 and/or human IL-13 activity with an IC₅₀ of about1×10¹¹M or less.

IL-13 exerts its actions by binding to the IL-13 receptor (IL-13R) onthe cell surface, the heterodimer comprised of the IL-13Rα1 chain(IL-13Rα1) and the IL-4R chain (IL-4R). IL-13 binds to IL-13Rα1 firstwith low affinity (KD=2⁻¹° nM) and then recruits IL-4R to the complex,generating a high affinity receptor (KD=0.03-0.4 nM) (Aman, M. J., etal. 1996 J. Biol. Chem. 271, 29265-29270; Miloux, et al. 1997 FEBS Lett.401, 163-166; Andrews, et al 2002 J. Biol. Chem. 277, 46073-46078).Heterodimerization of IL-13R causes activation of Janus kinases, TYK2and JAKE constitutively associated with IL-13Rα1 and IL-4R,respectively, followed by activation of the signal transducer andactivator of transcription 6 (STAT6) (Izuhara, K., and Arima, K. 2004Drug News Perspect. 17, 91-98). There is another IL-13-binding unit, theIL-13Rα2 chain (IL-13Rα2), which binds to IL-13 with high affinity(0.25-1.2 nM) (Caput, et al 1996 J. Biol. Chem. 271, 16921-16926;Donaldson et al 1998 J. Immunol. 161, 2317-2324). No other receptormolecule is known to be involved in the IL-131L-13R2 complex. IL-13R2 isinitially thought to act as a nonsignaling “decoy” receptor. However, itwas later discovered that it can bind to IL-13 and signals through AP-1pathway, leading to TNF-beta production in certain cell types includingmacrophages, which in turn leads to lung fibrosis (Fichtner-Feigl, 2006Nat Med 12:99-106). Therefore both IL-13Rα1/IL-4Rα and IL-13Rα2 pathwayscontribute to the overall pathophysiology of asthma and other pulmonaryinflammatory conditions. Therefore, a therapeutic anti-IL-13 antibodythat blocks IL-13 binding to both receptors will be more effective thatthose that blocks only one receptor.

In one aspect, the instant invention provides compositions and/ormethods which use monoclonal antibodies that block IL-13 binding to bothIL-13Rα1 and IL-13Rα2. Both ELISA-based receptor binding assay and125-1-labeled IL-13 binding assay on cell surface demonstrated that13C5, both murine version and humanized version (i.e., 13C5.5), wereable to effective block IL-13 binding to both receptors. Antibodies inthe same lineage as 13C5, including 25C8 and 33C3, were also able toblock IL-13 binding to both receptors. Epitope mapping of 13C5 indicatedthat its binding site(s) included the C-terminal Helix D region of humanIL-13 (residues VRDTK IEVAQ FVKDL LLHLK KLFRE GR, corresponding to aminoacid 104-130 of SEQ ID NO. 1). The c-terminal helix D region has beenproposed to be involved in interactions with the IL-13 receptor (Zuegget al 2001 Immunol Cell Biol. 79:332-9). Crystal structure of humanIL-13 complexed with the Fab portion of 13C5.5 antibody indicated that13C5.5 binds the C-terminal Helix D region as well as the N-terminalHelix A region of human IL-13. Preferably the antibody, or antigenbinding fragment thereof binds human IL-13 such to that IL-13 with saidantibody, or antigen binding fragment thereof, bound to the epitopedefined by the topographic regionsSer26-Thr27-Ala28-Leu29-Arg30-Glu31-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-A-sn38and Lys123-Lys124-Leu125-Phe126-Arg127-Glu-128-Gly129-Arg130 of SEQ IDNo. 1 is inhibited from binding to the IL-13 receptor. Preferably theantibody, or antigen binding fragment thereof binds human IL-13 suchthat IL-13 with said antibody, or antigen binding fragment thereof,bound to the epitope defined by the topographic regionsArg30-Glu3′-Leu32-Ile33-Glu34-Glu35-Leu36-Val37-Asn38 andLys123-Lys124-Leu125-Phe126-Arg127 of SEQ ID No. 1 is inhibited frombinding to the IL-13.alpha.2 receptor.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgG1 heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (Winter, et al. U.S. Pat. Nos.5,648,260; 5,624,821). The Fc portion of an antibody mediates severalimportant effector functions e.g. cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding toFc.gamma.Rs and complement C1q, respectively. Neonatal Fc receptors(FcRn) are the critical components determining the circulating half-lifeof antibodies. In still another embodiment at least one amino acidresidue is replaced in the constant region of the antibody, for examplethe Fc region of the antibody, such that effector functions of theantibody are altered.

In one embodiment, the methods and compositions of the invention use alabeled binding protein wherein an antibody or antibody portion of theinvention is derivatized or linked to another functional molecule (e.g.,another peptide or protein). For example, a labeled binding protein ofthe invention can be derived by functionally linking an antibody orantibody portion of the invention (by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody (e.g., a bispecific antibody or adiabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent,and/or a protein or peptide that can mediate associate of the antibodyor antibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

In another embodiment, the compositions and methods of the invention usea crystallized binding protein. Preferably the invention relates tocrystals of whole anti-IL-13 antibodies and fragments thereof asdisclosed herein, and formulations and compositions comprising suchcrystals. In one embodiment the crystallized binding protein has agreater half-life in vivo than the soluble counterpart of the bindingprotein. In another embodiment the binding protein retains biologicalactivity after crystallization.

Crystallized binding protein of the invention may be produced accordingmethods known in the art and as disclosed in WO 02072636, incorporatedherein by reference.

In yet another embodiment, the compositions and/or methods of theinvention use a glycosylated binding protein wherein the antibody orantigen-binding portion thereof comprises one or more carbohydrateresidues. Nascent in vivo protein production may undergo furtherprocessing, known as post-translational modification. In particular,sugar (glycosyl) residues may be added enzymatically, a process known asglycosylation. The resulting proteins bearing covalently linkedoligosaccharide side chains are known as glycosylated proteins orglycoproteins. Antibodies are glycoproteins with one or morecarbohydrate residues in the Fc domain, as well as the variable domain.Carbohydrate residues in the Fc domain have important effect on theeffector function of the Fc domain, with minimal effect on antigenbinding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21(2005), pp. 11-16). In contrast, glycosylation of the variable domainmay have an effect on the antigen binding activity of the antibody.Glycosylation in the variable domain may have a negative effect onantibody binding affinity, likely due to steric hindrance (Co, M. S., etal., Mol. Immunol. (1993) 30:1361-1367), or result in increased affinityfor the antigen (Wallick, S. C., et al., Exp. Med. (1988) 168:1099-1109;Wright, A., et al., EMBO J. (1991) 10:2717 2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies.Glycosylation site mutants that retain the biological activity, but haveincreased or decreased binding activity, are another object of thepresent invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified antibody of the invention canbe made that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S. patentapplications 20040018590 and 20020137134 and PCT publicationWO2005100584 A2, the entire contents of each of which are incorporatedby reference herein).

The methods and/or compositions of the present invention may also use ananti-idiotypic (anti-Id) antibody specific for such binding proteins ofthe invention. An anti-Id antibody is an antibody, which recognizesunique determinants generally associated with the antigen-binding regionof another antibody. The anti-Id can be prepared by immunizing an animalwith the binding protein or a CDR containing region thereof. Theimmunized animal will recognize, and respond to the idiotypicdeterminants of the immunizing antibody and produce an anti-Id antibody.The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein to having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

II. Compositions of the Invention

The invention also provides compositions, e.g., pharmaceuticalcompositions, comprising an antibody, or antigen-binding portionthereof, of the invention and a pharmaceutically acceptable carrier. Thecompositions of the invention comprise an anti-IL-13 antibody, orantigen-binding portion thereof, such that when administeredintravenously to a subject at a dose of about 0.3 mg/kg, the antibody,or antigen-binding portion thereof, is capable of exhibiting: (a) anarea under the curve (AUC) of between about 1,500 and about 2,700μgh/ml; (b) a volume of distribution of between about 65 and 125 mL/kg;(c) a peak concentration (C_(max)) of between about 5 and about 8 μg/ml;and (d) a clearance rate of between about 0.1 and about 0.2 ml/h/kg.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,such that, when administered intravenously to a subject at a dose ofabout 3 mg/kg, the antibody, or antigen-binding portion thereof, iscapable of exhibiting: (a) an area under the curve (AUC) of betweenabout 21,000 and about 33,500 μgh/ml; (b) a volume of distribution ofbetween about 55 and about 100 mL/kg; (c) a peak concentration (C_(max))of between about 55 and about 90 μg/ml; and (d) a clearance rate ofbetween about 0.08 and about 0.15 ml/h/kg.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,such that, when administered intravenously to a subject at a dose ofabout 10 mg/kg, the antibody, or antigen-binding portion thereof, iscapable of exhibiting: (a) an area under the curve (AUC) of betweenabout 75 and about 100 μgh/ml; (b) a volume of distribution of betweenabout 90 and about 130 mL/kg; (c) a peak concentration (C_(max)) ofbetween about 185 and about 250 μg/ml; and (d) a clearance rate ofbetween about 0.1 and about 0.15 ml/h/kg.

In yet another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,such that, when administered subcutaneously to a subject at a dose ofabout 0.3 mg/kg, the antibody, or antigen-binding portion thereof, iscapable of exhibiting: (a) an area under the curve (AUC) of betweenabout 125 and about 800 μgh/ml; and (b) a peak concentration (C_(max))of between about 1.0 and about 6.0 μg/ml.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,such that, when administered subcutaneously to a subject at a dose ofabout 3 mg/kg, the antibody, or antigen-binding portion thereof, iscapable of exhibiting: (a) an area under the curve (AUC) of betweenabout 1,100 and about 8,500 μgh/ml; and (b) a peak concentration(C_(max)) of between about 12 and about 60 μg/ml.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,such that, when administered intravenously to a subject at a dose ofabout 0.3 mg/kg, 1 mg/kg, 3 mg/kg or 10 mg/kg, the antibody, orantigen-binding portion thereof, is capable of exhibiting any of thepharmacokinetic parameters set forth in the specification, Tables orFigures.

In another aspect, the invention provides an isolated compositioncomprising an anti-IL-13 antibody, or antigen-binding portion thereof,such that, when administered subcutaneously to a subject at a dose ofabout 0.3 mg/kg, 1 mg/kg or 3 mg/kg, the antibody, or antigen-bindingportion thereof, is capable of exhibiting any of the pharmacokineticparameters set forth in the specification, Tables or Figures.

The pharmaceutical compositions comprising antibodies of the inventionare for use in, but not limited to, diagnosing, detecting, or monitoringa disorder, in preventing, treating, managing, or ameliorating of adisorder or one or more symptoms thereof, and/or in research. In aspecific embodiment, a composition comprises one or more antibodies ofthe invention. In another embodiment, the pharmaceutical compositioncomprises one or more antibodies of the invention and one or moreprophylactic or therapeutic agents other than antibodies of theinvention for treating a disorder in which IL-13 activity isdetrimental, such as asthma. Preferably, the prophylactic or therapeuticagents known to be useful for or having been or currently being used inthe prevention, treatment, management, or amelioration of a disorder orone or more symptoms thereof. In accordance with these embodiments, thecomposition may further comprise of a carrier, diluent or excipient.

Typically, a pharmaceutical composition of the invention comprises ananti-IL-13 antibody, or antigen-binding portion thereof, and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidurala administration, intratumoral administration,and mucosal administration (e.g., intranasal and oral routes). Inaddition, pulmonary administration can be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. See,e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos.WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903,each of which is incorporated herein by reference their to entireties.In one embodiment, an antibody of the invention, combination therapy, ora composition of the invention is administered using Alkermes AIR™pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer thecompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion, by injection, or by means of an implant, said implantbeing of a porous or non-porous material, including membranes andmatrices, such as sialastic membranes, polymers, fibrous matrices (e.g.,Tissuel™), or collagen matrices. In one embodiment, an effective amountof one or more antibodies of the invention antagonists is administeredlocally to the affected area to a subject to prevent, treat, manage,and/or ameliorate a disorder or a symptom thereof. In anotherembodiment, an effective amount of one or more antibodies of theinvention is administered locally to the affected area in combinationwith an effective amount of one or more therapies (e.g., one or moreprophylactic or therapeutic agents) other than an antibody of theinvention of a subject to prevent, treat, manage, and/or ameliorate adisorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent of theinvention can be delivered in a controlled release or sustained releasesystem. In one embodiment, a pump may be used to achieve controlled orsustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref.Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used to achieve controlled or sustained release of thetherapies of the invention (see e.g., Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983,J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,1985, Science 228:190; During et al., 1989, Ann Neurol. 25:351; Howardet al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos. 5,679,377;5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO99/15154; and PCT Publication No. WO 99/20253. Examples of polymers usedin sustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In apreferred embodiment, the polymer used in a sustained releaseformulation is inert, free of leachable impurities, stable on storage,sterile, and biodegradable. In yet another embodiment, a controlled orsustained release system can be placed in proximity of the prophylacticor therapeutic target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity preferablygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art.

For intranasal administration of a composition of the invention, thecomposition can be formulated in an aerosol form, spray, mist or in theform of drops. In particular, prophylactic or therapeutic agents for useaccording to the present invention can be conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant (e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

For oral administration, compositions of the invention can be formulatedorally in the form of tablets, capsules, cachets, gelcaps, solutions,suspensions, and the like. Tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone,or hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose, or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well-known in theart. Liquid preparations for oral administration may take the form of,but not limited to, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives, orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, or fractionated vegetable oils); and preservatives (e.g.,methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparationsmay also contain buffer salts, flavoring, coloring, and sweeteningagents as appropriate. Preparations for oral administration may besuitably formulated for slow release, controlled release, or sustainedrelease of a prophylactic or therapeutic agent(s).

The methods of the invention may comprise pulmonary administration,e.g., by use of an inhaler or nebulizer, of a composition formulatedwith an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968,5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO97/44013, WO 98/31346, and WO 99/66903, each of which is incorporatedherein by reference their entireties. In a specific embodiment, anantibody of the invention, combination therapy, and/or composition ofthe invention is administered using Alkermes AIR™ pulmonary drugdelivery technology (Alkermes, Inc., Cambridge, Mass.).

The methods of the invention may also comprise administration of acomposition formulated for parenteral administration by injection (e.g.,by bolus injection or continuous infusion). Formulations for injectionmay be presented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompasses administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention may be packaged in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof the agent. In one embodiment, the compositions of the invention maybe supplied as a dry sterilized lyophilized powder or water freeconcentrate in a hermetically sealed container and can be reconstituted(e.g., with water or saline) to the appropriate concentration foradministration to a subject. Preferably, the compositions of theinvention are supplied as a dry sterile lyophilized powder in ahermetically sealed container at a unit dosage of at least 5 mg, morepreferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg.The lyophilized prophylactic or therapeutic agents or pharmaceuticalcompositions of the invention should be stored at between 2° C. and 8°C. in its original container and the prophylactic or therapeutic agents,or pharmaceutical compositions of the invention should be administeredwithin 1 week, preferably within 5 days, within 72 hours, within 48hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours,within 3 hours, or within 1 hour after being reconstituted. In analternative embodiment, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied inliquid form in a hermetically sealed container indicating the quantityand concentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml or at least 100 mg/ml. The liquid form should be storedat between 2° C. and 8° C. in its original container.

Compositions of the invention are preferably suitable for parenteraladministration. For example, the compositions of the invention may beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 24%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-Methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants. The pharmaceutical composition comprising theantibodies and antibody-portions of the invention prepared as aninjectable solution for parenteral administration, can further comprisean agent useful as an adjuvant, such as those used to increase theabsorption, or dispersion of a therapeutic protein (e.g., antibody). Aparticularly useful adjuvant is hyaluronidase, such as Hylenex™(recombinant human hyaluronidase). Addition of hyaluronidase in theinjectable solution improves human bioavailability following parenteraladministration, particularly subcutaneous administration. It also allowsfor greater injection site volumes (i.e. greater than 1 ml) with lesspain and discomfort, and minimum incidence of injection site reactions.(see WO2004078140, US2006104968 incorporated herein by reference).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The compositions of the present invention can be administered by avariety of methods known in the art, although for many therapeuticapplications, the preferred route/mode of administration is subcutaneousinjection, intravenous injection or infusion. As will be appreciated bythe skilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, the compositions of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell to gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingdisorders in which IL-13 activity is detrimental. For example, ananti-IL-13 antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind othercytokines or that bind cell surface molecules). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

In certain embodiments, an anti-1′-13 antibody, or antigen-bindingportion thereof, is linked to a half-life extending vehicle known in theart. Such vehicles include, but are not limited to, the Fc domain,polyethylene glycol, and dextran. Such vehicles are described, e.g., inU.S. application Ser. No. 09/428,082 and published PCT Application No.WO 99/25044, which are hereby incorporated by reference for any purpose.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. In oneembodiment, a therapeutically or prophylactically effective amount ofthe antibody, or antigen-binding portion thereof, is 0.3 mg/kg. Inanother embodiment, a therapeutically or prophylactically effectiveamount of the antibody, or antigen-binding portion thereof, is 3 mg/kg.In another embodiment, a therapeutically or prophylactically effectiveamount of the antibody, or antigen-binding portion thereof, is 10 mg/kg.In yet another embodiment, a therapeutically or prophylacticallyeffective amount of the antibody, or antigen-binding portion thereof, is0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.25 mg/kg, 1.5 mg/kg, 1.75 mg/kg,2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9mg/kg, 9.5 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg or 20 mg/kg. It is to benoted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

III. Methods of the Invention

In another aspect, this application features a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing anIL-13-associated disorder, such as asthma, in a subject. The methodincludes: administering to the subject an IL-13 binding agent(particularly an antagonist), e.g., an anti-IL-13 antibody orantigen-biding portion thereof as described herein, in an amountsufficient to treat or prevent the IL-13-associated disorder, such asasthma. The IL-13 antagonist, e.g., the anti-IL-13 antibody orantigen-binding portion thereof, can be administered to the subject,alone or in combination with other therapeutic modalities as describedherein.

In one embodiment, the subject is a mammal, e.g., a human suffering fromone or more IL-13-associated disorders, including, e.g., respiratorydisorders (e.g., asthma (e.g., allergic and nonallergic asthma or mildasthma, or moderate asthma), chronic obstructive pulmonary disease(COPD), and other conditions involving airway inflammation,eosinophilia, fibrosis and excess mucus production; atopic disorders(e.g., atopic dermatitis and allergic rhinitis). Accordingly, thedisclosure includes the use of an IL-13 binding agent (such as ananti-IL-13 antibody or fragment thereof described herein) for atreatment described herein and the use of an IL-13 binding agent (suchas an anti-IL-13 antibody or fragment thereof described herein) forpreparing a medicament for a treatment described herein.

Examples of IL-13-associated disorders include, but are not limited to,a disorder chosen from one or more of: respiratory disorders, e.g.,asthma (e.g., allergic and nonallergic asthma (e.g., asthma due toinfection with, e.g., respiratory syncytial virus (RSV), e.g., inyounger children)), chronic obstructive pulmonary disease (COPD), andother conditions involving airway inflammation, eosinophilia, fibrosisand excess mucus production, e.g., cystic fibrosis and pulmonaryfibrosis.

In other embodiments, this application provides a method of treating(e.g., reducing, ameliorating) or preventing one or more symptomsassociated with a respiratory disorder, e.g., asthma (e.g., allergic andnonallergic asthma); allergies;

chronic obstructive pulmonary disease (COPD); a condition involvingairway inflammation, eosinophilia, fibrosis and excess mucus production,e.g., cystic fibrosis and pulmonary fibrosis. For example, symptoms ofasthma include, but are not limited to, wheezing, shortness of breath,bronchoconstriction, airway hyperreactivity, decreased lung capacity,fibrosis, airway inflammation, and mucus production. The methodcomprises administering to the subject an IL-13 antagonist, e.g., anIL-13 antibody or a fragment thereof, in an amount sufficient to treat(e.g., reduce, ameliorate) or prevent one or more symptoms. The IL-13antibody can be administered therapeutically or prophylactically, orboth. The IL-13 antagonist, e.g., the anti-IL-13 antibody, orantigen-binding portion thereof, can be administered to the subject,alone or in combination with other therapeutic modalities as describedherein. Preferably, the subject is a mammal, e.g., a human sufferingfrom an IL-13-associated disorder, such as asthma, as described herein.

In another aspect, this application provides a method for detecting thepresence of IL-13 in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, biopsy). The subject method can be used todiagnose a disorder, e.g., asthma, e.g., mild or moderate asthma. Themethod includes: (i) contacting the sample or a control sample with theanti-IL-13 antibody or fragment thereof as described herein; and (ii)detecting formation of a complex between the anti-IL-13 antibody orfragment thereof, and the sample or the control sample, wherein astatistically significant change in the formation of the complex in thesample relative to the control sample is indicative of the presence ofthe IL-13 in the sample.

In yet another aspect, this application provides a method for detectingthe presence of IL-13 in vivo (e.g., in vivo imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., anIL-13-associated disorder, e.g., asthma, e.g., mild or moderate asthma.The method includes: (i) administering the anti-IL-13 antibody orfragment thereof as described herein to a subject or a control subjectunder conditions that allow binding of the antibody or fragment toIL-13; and (ii) detecting formation of a complex between the antibody orfragment and IL-13, wherein a statistically significant change in theformation of the complex in the subject relative to the control subjectis indicative of the presence of IL-13.

Antibodies of the invention, or antigen binding portions thereof can beused alone or in combination to treat such diseases. It should beunderstood that the antibodies of the invention or antigen bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agent thatimparts a beneficial attribute to the therapeutic composition e.g., anagent which effects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The combination therapy can include one or more IL-13 antagonists, e.g.,anti-IL-13 antibodies or fragments thereof, coformulated with, and/orcoadministered with, one or more additional therapeutic agents, e.g.,one or more cytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents (e.g., systemic anti-inflammatory agents),anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors, and/orcytotoxic or cytostatic agents, as described in more herein.

Examples of preferred additional therapeutic agents that can becoadministered and/or coformulated with one or more IL-13 antagonists,e.g., anti-IL-13 antibodies or fragments thereof, include, but are notlimited to, one or more of: inhaled steroids; beta-agonists, e.g.,short-acting or long-acting beta-agonists; antagonists of leukotrienesor leukotriene receptors; combination drugs such as ADVAIR; IgEinhibitors, e.g., anti-IgE antibodies (e.g., XOLAIR); phosphodiesteraseinhibitors (e.g., PDE4 inhibitors); xanthines; anticholinergic drugs;mast cell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or itsreceptors including H1, H2, H3, and H4, and antagonists of prostaglandinD or its receptors (DP1 and CRTH2). Such combinations can be used totreat asthma and other respiratory disorders. Additional examples oftherapeutic agents that can be coadministered and/or coformulated withone or more anti-IL-13 antibodies or fragments thereof include one ormore of: TNF antagonists (e.g., a soluble fragment of a TNF receptor,e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kDTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL)); TNF enzymeantagonists, e.g., TNF converting enzyme (TACE) inhibitors; muscarinicreceptor antagonists; TGF-beta antagonists; interferon gamma;perfenidone; chemotherapeutic agents, e.g., methotrexate, leflunomide,or a sirolimus (rapamycin) or an analog thereof, e.g., CCI-779; COX2 andcPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors, TPL-2, MK-2and NFkB inhibitors, among others.

Other preferred combinations are cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-31, interferons, EMAP-II,GM-CSF, FGF, EGF, PDGF, and edothelin-1, as well as the receptors ofthese cytokines and growth factors. Antibodies of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligandsincluding CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the inflammatory cascade; preferred examples include TNFantagonists like chimeric, humanized or human TNF antibodies, D2E7, (PCTPublication No. WO 97/29131), CA2 (Remicade™), CDP 571, and soluble p55or p75 TNF receptors, derivatives, thereof, (p75TNFR1gG (Enbrel™) orp55TNFR IgG (Lenercept), and also TNF converting enzyme (TACE)inhibitors; similarly IL-1 inhibitors (Interleukin-1-converting enzymeinhibitors, IL-1RA etc.) may be effective for the same reason. Otherpreferred combinations include Interleukin 4. Yet another preferredcombination are other key players of the asthmatic response which mayact parallel to, dependent on or in concert with IL-13 function;especially preferred are IL-9 antagonists including IL-9 antibodies. Ithas been shown that IL-13 and IL-9 have overlapping but distinctfunctions and a combination of antagonists to both may be mosteffective. Yet another preferred combination are anti-IL-5 antibodies.Yet other preferred combinations include antagonists of chemokinesincluding MCP-1, MCP-4, eotaxins, RANTES, MDC, CCL-12 and CCL-17 (TARC)and chemokine receptors including CCR2, CCR3, CCR4, and CXCR4. Yetcombinations can include antagonists to asthma mediators including acidmammalian chitinase, CRHT2, chymase, S1P1, S1P2, Tyk2, ROCKII, Stat6,p38, NFkB, phosphodiesterase 4 (PDE-4), mast cell trytase, NO,adenosine, IKK2, GATA3, ICAM-1, VCAM-1, and ICOS.

As used herein, the term “a disorder in which IL-13 activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of IL-13 in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. In one embodiment, the disorder in whichIL-13 activity is detrimental is asthma, e.g., mild asthma or moderateasthma. Accordingly, a disorder in which IL-13 activity is detrimentalis a disorder in which reduction of IL-13 activity is expected toalleviate the symptoms and/or progression of the disorder. Suchdisorders may be evidenced, for example, by an increase in theconcentration of IL-13 in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of IL-13 in serum,plasma, synovial fluid, etc. of the subject), which can be detected, forexample, using an anti-IL-13 antibody as described above. Non-limitingexamples of disorders that can be treated with the antibodies of theinvention include asthma, e.g., mild or moderate asthma, as well asthose disorders discussed in the section below pertaining topharmaceutical compositions of the antibodies of the invention.

IL-13 has been implicated as having a pivotal role in causingpathological responses associated with asthma. However other mediatorsof differential immunological pathways are also involved in asthmapathogenesis, and blocking these mediators, in addition to IL-13, mayoffer additional therapeutic benefit. Thus, binding proteins of theinvention may be incorporated into DVD-Ig proteins where in the DVD iscapable of binding target pairs including, but not limited to, IL-13 anda pro-inflammatory cytokine, such as tumor necrosis factor-α (TNF-α).TNF-α may amplify the inflammatory response in asthma and may be linkedto disease severity (McDonnell, et al., Progress in Respiratory Research(2001), 31(New Drugs for Asthma, Allergy and COPD), 247-250). Thissuggests that blocking both IL-13 and TNF-α may have beneficial effects,particularly in severe airway disease. In a preferred embodiment theDVD-Ig of the invention binds the targets IL-13 and TNFα and is used fortreating asthma.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references, including literature references, issued patents,and published patent applications, as cited throughout this applicationare hereby expressly incorporated herein by reference. It should furtherbe understood that the contents of all the figures and tables attachedhereto, as well as the entire contents of U.S. Pat. No. 7,915,388 areexpressly incorporated herein by reference.

EXAMPLES Introduction to Examples

Asthma is a chronic inflammatory disorder of the airways characterizedby wheezing, breathlessness, chest tightness, and cough. Asthma affectsapproximately 20 million people in the US, and about 75% of asthmapatients are adults. Of the adult asthma patients, approximately 60% ofasthma patients have mild disease, about 20% have moderate disease andthe remaining 20% have severe disease.

Interleukin-13 (IL-13) is thought to be pivotal in the pathogenesis ofhuman asthma, in that elevated levels of IL-13 are present in the lungsof asthma patients, and these levels correlate with disease severity(FIG. 1). Likewise, increased IL-13 is present in both sputum and inlung biopsies of patients with moderate to severe asthma who are treatedwith inhaled corticosteroids (ICS) or systemic corticosteroids andcontinue to be symptomatic. Moreover, human IL-13 genetic polymorphismsare associated with asthma and atopy (allergic hypersensitivity). IL-13binds to two receptors, IL-13Rα1 and IL-13Rα2. IL-13 is a well-validatedtarget for asthma as efficacy has been demonstrated using various meansof IL-13 antagonism in multiple, pre-clinical models of asthma.

13C5.5 is a humanized recombinant immunoglobulin IgG1 (IgG1, κ)monoclonal antibody (mAb) specific for human wild-type and variantIL-13. 13C5.5 recognizes a unique epitope on IL-13 that blocks itsbinding to both IL-13 receptors α1 and α2 (FIG. 2); other similarIL-13-binding antibodies analyzed to date only block the α1 receptor asdetermined by crystallography and biochemical characterization. 13C5.5is selective for IL-13, and does not recognize other cytokines. Tworesidues (L240A and L241A) in the heavy chain were mutated to prevent Fcgamma receptor and complement binding. Likewise, 13C5.5 does not bind orstimulate human whole blood cells to release cytokines. The heavy andlight chain variable regions are set forth above as SEQ ID NO:2 and SEQID NO:3, respectively.

13C5.5 binds both human wild type and variant IL-13 with high affinityand potency; this is important because variant IL-13 is present in ˜20%of human asthmatics. 13C5.5 does not cross-react with mouse, sheep, ordog IL-13. 13C5.5 does cross-react with cynomolgus monkey rIL-13 with a51 fold lower in vitro potency and with rat rIL-13 with a 155 fold lowerin vitro potency compared to human rIL-13. Although the affinity andpotency of 13C5.5 is lower against cynomolgus and rat rIL-13 in vitro,13C5.5 fully neutralizes cynomolgus monkey rIL-13 in vitro with a halfmaximal inhibitory concentration (IC₅₀) of 4.1 nM and rat IL-13 with anIC₅₀ of 12.4 nM.

Initial pharmacokinetic analysis following single-dose administration of13C5.5 was conducted in Sprague-Dawley rats and cynomolgus monkey (FIG.3). 13C5.5 generally exhibited low clearance, low volume ofdistribution, and high bioavailability. The half-life varied fromapproximately 12 to 16 days in rats and 6 to 11 days in monkeys. Thepharmacokinetics of 13C5.5 was generally similar between female and malemonkeys and rats.

13C5.5 was well tolerated in 4-week rat and 2-week monkey toxicologystudies and had sufficient safety margins relative to the recommendedstarting dose for human clinical trials. Theno-observed-adverse-effect-levels (NOAELs) during the repeat dosetoxicology studies were 1500 mg/kg/dose via intravenous (IV)administration in rats and cynomolgus monkey and 200 mg/kg/dose viasubcutaneous (SC) administration in cynomologus monkey.

This study was a Phase 1 first-in-human study in adult subjects with orwithout mild to moderate controlled asthma. This placebo-controlledstudy evaluated 4 single escalating doses (0.3, 1.0, 3.0, and 10.0mg/kg) of 13C5.5 administered IV and 2 doses (0.3 and 3.0 mg/kg) of13C5.5 administered as 3 weekly SC doses.

A study of 13C5.5 conducted in healthy subjects and subjects withmild-to-moderate controlled asthma allowed for collection of standarddata regarding human pharmacokinetics and bioavailability of 13C5.5 aswell as the tolerability, safety, and immunogenicity with single- andmultiple-escalating doses of 13C5.5 in the target disease indication.

Experimental Protocol

This was a Phase 1, single and multiple escalating dose,placebo-controlled, double-blind, randomized, 3-part study which wasconducted according to a sequential design. Adults in general goodhealth (n=20) and adults with mild to moderate controlled asthma (n=27)were selected to participate in the study according to the selectioncriteria. No more than one subject was dosed per cohort per day.

Part 1 of the study consisted of four groups (Groups 1 through 4), with5 subjects in each group. For Part 1, after meeting the selectioncriteria, adults in general good health (n=20) were assigned to one ofthe following four single dose groups:

-   -   Group 1 (intravenous infusion of 0.3 mg/kg 13C5.5 or placebo),    -   Group 2 (intravenous infusion of 1.0 mg/kg 13C5.5 or placebo),    -   Group 3 (intravenous infusion of 3.0 mg/kg 13C5.5 or placebo),        or    -   Group 4 (intravenous infusion of 10.0 mg/kg 13C5.5 or placebo).

Within each group, four subjects were randomized to receive 13C5.5 andone subject was randomized to receive placebo. Dose escalation tookplace for a new cohort after all subjects within a dose cohort hadsatisfactorily completed at least the minimum one-week safetyassessments.

Part 2 of the study consisted of three groups (Groups 5 through 7), with5 subjects randomized to each of Groups 5, 6 and 7. For Part 2, aftermeeting the selection criteria, adult subjects with mild-to-moderatecontrolled asthma (n=15) were assigned to one of three single dosegroups:

-   -   Group 5 (intravenous infusion of 0.3 mg/kg 13C5.5 or placebo),    -   Group 6 (intravenous infusion of 3.0 mg/kg 13C5.5 or placebo),        or    -   Group 7 (intravenous infusion of 10.0 mg/kg 13C5.5 or placebo).

Within Groups 5, 6 and 7, four subjects were randomized to receive13C5.5 and one subject was randomized to receive placebo.

As shown in FIG. 4, dosing for a cohort in Part 2 was allowed to beginafter all subjects of the same dose level in Part 1 and all lower doselevels in Part 2 had satisfactorily completed at least the minimumone-week safety assessments after the last dose.

Part 3 of the study consisted of two groups (Groups 8 and 9), with 6subjects randomized to each of Groups 8 and 9. For Part 3, after meetingthe selection criteria, adult subjects with mild to moderate controlledasthma (n=12) were assigned to one of two groups:

-   -   Group 8 (subcutaneous injection of 0.3 mg/kg 13C5.5 or placebo,        three weekly doses) or    -   Group 9 (subcutaneous injection of 3.0 mg/kg 13C5.5 or placebo,        three weekly doses).

Within Groups 8 and 9, four subjects were randomized to receive 13C5.5and two subjects were randomized to receive placebo.

Dosing for cohorts in Part 3 was allowed to begin after all subjectswithin the same dose level in Part 2, and one dose level higher in Part1 and all lower dose levels in Part 3 had satisfactorily completed atleast the minimum one-week safety assessments after the last dose (FIG.4).

The study was conducted in a double-blind manner such that theinvestigators and subjects were blinded to treatment assignments withineach group. For evaluation of safety, the medical monitor was unblindedto treatment assignments. A diagram of the treatment groups is shown inTable 1.

TABLE 1 Treatment Groups Number of Part Group Regimen Subjects Part 1*Group 1 0.3 mg/kg 13C5.5 IV or 5 (4:1) Healthy Subjects placebo Group 21.0 mg/kg 13C5.5 IV or 5 (4:1) placebo Group 3 3.0 mg/kg 13C5.5 IV or 5(4:1) placebo Group 4 10.0 mg/kg 13C5.5 IV or 5 (4:1) placebo Part2*^(#) Group 5 0.3 mg/kg 13C5.5 IV or 5 (4:1) Mild/Moderate placeboControlled Group 6 3.0 mg/kg 13C5.5 IV or 5 (4:1) Asthma placebo Group 710.0 mg/kg 13C5.5 IV or 5 (4:1) placebo Part 3*^($) Group 8 0.3 mg/kg13C5.5 SC or 6 (4:2) Mild/Moderate placebo, Controlled 3 weekly dosesAsthma Group 9 3.0 mg/kg 13C5.5 SC or 6 (4:2) placebo, 3 weekly doses*Dose escalation took place for a new cohort after all subjects within acohort had satisfactorily completed at least the minimum one-week safetyassessments. ^(#)Group 5 was dosed after completion of Group 1 andevaluation of adequate safety for that dosing cohort (minimum one-weeksafety assessments). A similar approach was taken with Group 6 aftercompletion of Groups 3 and 5 and Group 7 after completion of Groups 4and 6. ^($)Group 8 was dosed after completion of Groups 2 and 5 andevaluation of adequate safety for those dosing cohorts and evaluation ofadequate safety (minimum one-week safety assessments). A similarapproach was taken with Group 9 after completion of Groups 4, 6 and 8.

For Parts 1 and 2, study drug or placebo was administered on Study Day 1for each dose group. For Part 3, study drug or placebo was administeredon Study Days 1, 8, and 15 for each dose group. Study drugadministration on Study Days 8 and 15 may have occurred within a 24-hourperiod of the scheduled dose in cohorts 8 and 9. A minimum of one weekseparated the different dose levels.

Subjects were confined to the study site and supervised forapproximately 4 to 6 days if they were in an IV dose group (Parts 1 and2) or approximately 6 to 8 days if they were in an SC dose group (Part3). Confinement began in the afternoon, of the Baseline day which mayhave occurred anytime within a 3-day window (i.e., Study Day −3 to StudyDay −1) prior to dosing on Study Day 1, and ended after the collectionof the 48-hour blood samples (Study Day 3) for subject's enrolled in theIV dosing groups, or after the collection of the 96-hour blood sample(Study Day 5) for subject's enrolled in the SC dosing groups. Strenuousactivity during confinement was not permitted.

In addition, subjects in Part 3 were confined to the study site on StudyDays 8 and 15 for 24 hours. During confinement subjects received an SCdose of either 13C5.5 or placebo and were evaluated for vital signs,safety assessments, laboratory assessments, pharmacokinetics and ADA,Biomarker sampling, electrocardiograms (ECGs), and pulmonary functiontests (PFTs).

Subjects received a standardized diet, providing approximately 30% ofthe daily calories from fat, no more than 45% of daily calories fromcarbohydrates, and providing approximately 1900 calories/day for allmeals during confinement. The composition (protein, fat, carbohydrate,and total calories) of all meals was determined by a dietician, and arecord was kept with the source documents. During confinement in eachpart of the study, the subjects consumed only the scheduled mealsprovided in the study and water to quench thirst. The subjects abstainedfrom all other food and beverage.

For Parts 1 and 2, no food or beverage, except for water to quenchthirst, was allowed on Study Day −1 from 8 hours prior to dosing untilapproximately 2 hours after completion of the dosing.

For Part 3, no food or beverage, except for water to quench thirst, wasallowed on Study Days 1, 8, and 15 from 8 hours prior to dosing untilapproximately 2 hours after completion of the dosing on Study Day 1.

Inclusion Criteria

A subject was eligible for study participation if he/she met thefollowing criteria:

-   -   1. Males or females and age was between 18 and 55 years,        inclusive.    -   2. For Parts 2 and 3, a diagnosis of asthma for at least 6        months prior to Screening:        -   Mild to moderate controlled asthmatic subjects as defined            below            -   Use of prescribed short acting beta agonists for no more                than 4 puffs/week for symptomatic control of asthma            -   Long acting beta agonists (LABA) may have been taken as                prescribed if used concurrently with inhaled                corticosteroids (ICS). LABA was not administered for 12                hours before the screening PFTs were performed and could                resume at the completion of the testing            -   If on ICS, up to a medium dose may have been used                (defined as the equivalent of fluticasone MDS/HFA up to                440 mcg daily or fluticasone DPT up to 500 mcg daily)    -   3. For Parts 2 and 3, forced expiratory volume 1 (FEV₁)≧70% at        the Screening and Baseline visits.    -   4. For Parts 2 and 3, if on an ICS, a stable ICS dose for ≧4        weeks before Study Day 1 and dose was expected to remain stable        throughout the study. Must have used ICS for at least 4 weeks        before the screening visit.    -   5. For Parts 2 and 3, subjects with asthma had a positive        methacholine challenge test result available by history (within        the past 12 months) or demonstrated airway reversibility in        pulmonary function measurements by demonstrating at least 12%        increase in FEV₁ from best effort when tested at least 30        minutes after two to four inhalations of inhaled albuterol or        nebulized albuterol (or equivalent short acting beta agonist).        Up to 2 attempts could have been made at any one session to        demonstrate reversibility. Subjects may have returned on another        day for repeat testing if necessary. Reversibility or        methacholine challenge test confirming reversibility may have        been documented by medical record if performed within the past        12 months. A positive methacholine challenge was defined as a        PC₂₀ at a dose of methacholine of ≦8.0 mg/mL using American        Thoracic Society standards for pulmonary function testing (see        Guidelines for Methacholine and Exercise Challenge Testing 1999.        Am J. Respir. Crit. Care Med., Vol. 161, pp. 309-329, 2000).    -   6. For Parts 2 and 3, subjects had well-controlled asthma for at        least 4 weeks prior to Study Day 1, as defined by:        -   Asthma symptoms ≦2 days/week (i.e., cough, wheezing,            shortness of breath)        -   Nighttime awakenings ≦2 times during the prior 4-week period        -   No interference with normal activity        -   No more than 4 inhalations/week of a short acting beta            agonists for asthma symptom control (excluding the            prophylactic use for the prevention of exercise induced            bronchospasm).    -   7. If female, the subject met one of the following criteria:        -   Postmenopausal for at least two years,        -   Surgically sterile (bilateral oophorectomy, bilateral tubal            ligation, or hysterectomy). Females who had undergone tubal            ligation were required to agree to use a second form of            contraception starting on the first day of confinement until            160 days after study drug treatment which included:            -   Intrauterine (IUD) devices            -   Bather methods (diaphragm with spermicide, or condom                with spermicide)            -   Injected, oral, transdermal, vaginal, or implanted                methods of hormonal contraceptives    -   8. Females had negative results for pregnancy tests performed:    -   At Screening on a serum specimen obtained within 28 days prior        to Study Day 1 and at Baseline, which may have occurred anytime        within a 3 day window (i.e., Study Day −3 to Study Day −1) prior        to dosing Study Day 1.    -   9. If male, the subject was surgically sterile or practicing at        least 1 of the following methods of birth control during the        study and for 160 days after last study drug administration:        -   Subject used condom and partner(s) used an IUD        -   Subject used condom and partner(s) used oral, injected, or            implanted methods of hormonal contraceptives        -   Subject used condoms and partner(s) used bather method            (contraceptive sponge, diaphragm, or vaginal ring with            spermicidal jellies or creams)    -   Additionally, male subjects agreed to not donate sperm during        the study and for 160 days after the last dose of study drug.    -   10. For Part 1, Body Mass Index (BMI) was 18 to 29, inclusive.        For Parts 2 and 3, BMI was 18 to 34, inclusive. BMI was        calculated as weight in kg divided by square of height in        meters. Body weight did not exceed 120 kg.    -   11. A condition of general good health (other than mild-moderate        controlled asthma and associated medical conditions such as        mild-moderate allergic rhinitis, atopic dermatitis, and        gastroesophageal reflux disease) based upon the results of a        medical history, physical examination, vital signs, laboratory        profile and a 12-lead electrocardiogram (ECG).    -   12. Voluntarily signed and dated an informed consent approved by        an IRB, prior to the conduct of any screening or study-specific        procedures.

Exclusion Criteria

A subject was not eligible for study participation if he/she met any ofthe following criteria:

-   -   1. Subjects was using LABA therapy without concurrent use of        ICS.    -   2. For Parts 2 and 3, asthma exacerbation within 8 weeks of        Study Day 1.    -   3. For Parts 2 and 3, upper respiratory tract infection within 4        weeks of Study Day 1.    -   4. For Parts 2 and 3, asthma exacerbation requiring systemic        corticosteroids or any other reason for requiring systemic        corticosteroids within 6 months of Study Day 1.    -   5. For Parts 2 and 3, asthma exacerbation requiring emergency        room (ER) visit, hospitalization, or medical intervention within        6 months of Study Day 1.    -   6. History of clinically significant allergic reaction i.e.        anaphylaxis (per the investigator) to any drug, biologic, food        or vaccine.    -   7. History of major immunologic reaction to any IgG containing        agent.    -   8. History of atopic dermatitis involving ≧10% of body surface        area or requiring medical treatment other than use of low to        medium potency topical corticosteroids or over-the-counter        emollients within 6 months of Study Day 1.    -   9. History of diabetes (Type I or Type II) or a fasting serum        glucose level suggestive of diabetes (fasting serum glucose ≧126        mg/dL) at Screening.    -   10. History of an allergic reaction or significant sensitivity        to constituents of study drug.    -   11. History of tuberculosis (TB) or listeriosis.    -   12. History of persistent chronic or active infections which        required hospitalization or treatment with IV antibiotics, IV        antivirals or IV antifungals within 30 days of Screening or oral        antibiotics/antivirals within 14 days prior to Study Day 1.    -   13. Subjects were evaluated for latent TB infections. Subjects        demonstrated absence of TB infection or exposure as evidenced by        a negative chest X-ray and negative purified protein derivative        (PPD) skin test.    -   14. Positive test result for hepatitis B surface antigen        (HBsAg), hepatitis C virus antibody (HCV Ab), or HIV antibodies        (HIV Ab).    -   15. Positive test result on schistosomiasis serology.    -   16. History of unexplained diarrhea or abdominal pain of greater        than 2 weeks duration.    -   17. History of untreated parasitic infections.    -   18. History of genetic or acquired immune deficiency.    -   19. For Parts 2 and 3, use of any non-essential medications,        vitamins and/or herbal supplements within the 2-week period        prior to study drug administration. For Part 1, used any        prescription, over-the-counter medications or herbal supplements        within 2 weeks prior to study drug administration.    -   20. For Parts 2 and 3, subject had taken Xolair® within 5 months        of Study Day 1.    -   21. For Parts 2 and 3, subject had a change in immunotherapy        dose in the 3 months prior to Baseline.    -   22. Received any drug by injection within 30 days or 5        half-lives (whichever was longer) prior to study drug        administration.    -   23. Received any investigational product within 30 days or 5        half-lives (whichever was longer) prior to study drug        administration.    -   24. History of cancer or lymphoproliferative disease other than        a successfully treated non-metastatic cutaneous squamous cell or        basal cell carcinoma.    -   25. History of epilepsy, any clinically significant cardiac,        respiratory (except mild to moderate asthma), renal, hepatic,        gastrointestinal, hematologic, rheumatologic, or psychiatric        disease or disorder, non-healing wounds or recurrent poor wound        healing, or any uncontrolled medical illness. In addition the        following were excluded:        -   Chronic obstructive pulmonary disease (COPD), congestive            heart failure, pulmonary embolism, pulmonary infiltration            with eosinophilia, current cough secondary to drugs, vocal            cord dysfunctions.    -   26. History of cardiopulmonary Sudden Death in any first-degree        relative.    -   27. Pregnant or breast-feeding female.    -   28. Recent (6-month) history of drug or alcohol abuse.    -   29. Received of any live vaccine within 3 months prior to study        drug administration.    -   30. Positive screen for drugs of abuse, alcohol, or cotinine at        Screening or on admission.    -   31. Chest X-ray at Screening indicating any clinically        significant abnormality (including calcified granuloma and/or        pleural scarring) as assessed by appropriate medical personnel.    -   32. Febrile illness within 14 days prior to dosing.    -   33. Subjects with baseline QTc interval by Friderica correction        (QTcF)>450 msec for females and >430 msec for males.    -   34. Donated or lost a significant blood volume (including        plasmapheresis) or received a transfusion of any blood product        within 8 weeks prior to study drug administration.    -   35. Subject was a smoker, or had a history of smoking within the        6-month period preceding study drug administration.    -   36. Current enrollment in another clinical study.    -   37. Previous enrollment in this study.    -   38. Considered by the investigator or medical monitor, for any        reason, to be an unsuitable candidate to receive 13C5.5.

Treatments Administered

For Parts 1 and 2, a single dose of 13C5.5 or 13C5.5 placebo (0.3, 1.0,3.0, or 10.0 mg/kg) was administered intravenously to each subject inthe morning on Study Day 1. For Part 3, a total of 3 doses of 13C5.5 or13C5.5 placebo (0.3 or 3.0 mg/kg) were administered subcutaneously inthe morning on Study Days 1, 8, and 15. For the IV infusion, anindwelling catheter was inserted into a vein prior to dosing and wasflushed with 1 mL of 13C5.5 placebo. 13C5.5 or 13C5.5 placebo wasadministered intravenously by continuous infusion over approximately 120minutes with the subjects in a supine position. Following the infusion,a 1 mL 13C5.5 placebo flush was administered, and the line wasmaintained for a minimum of 2 hours following the completion of theinfusion with a 0.9% isotonic saline solution. Subjects remained in asupine position for at least 5 minutes before infusion and until 30minutes after the end of infusion.

For the SC dose, the study drug was administered subcutaneously into theleft upper quadrant of the abdomen, avoiding any blood vessels,thickening or tenderness of skin, scars, fibrous tissue, stretch marks,bruising, redness, nevi, or other skin imperfections. The subjectremained in a supine position for at least 30 minutes following eachinjection.

Subjects were assigned to one of nine dose groups (Table 2). Within eachIV infusion group, four subjects were randomized to receive 13C5.5 andone subject received placebo. Within each SC group, four subjectsreceived 13C5.5 and two received placebo. Dose escalation took place fora new cohort only after all subjects within a dose cohort hadsatisfactorily completed the minimum one-week safety assessments.

TABLE 2 Investigational Product: 13C5.5 Formulation 13C5.5 13C5.5Placebo Dosage Form Parenteral Parenteral Formulation Solution forSolution for injection in PFS injection in PFS Strength (mg) 100 mg per1.0 mL N/A Mode of Administration IV Infusion or SC IV Infusion or SCinjection injection Bulk Product Lot Number 09-023042 09-023043Manufacturing Site Germany Germany Finishing lot 09-024016 09-024017Retest Date 31 Jan. 2011 31 Jan. 2011 PFS = pre-filled syringe; N/A =Not applicable.

For IV dosing, study medication in the PFS was further diluted with13C5.5 placebo and mixed in an injection syringe for administration. ForSC dosing, no dilution was required, however material was transferred toinjection syringes for administration. The study drug was stored at 2°to 8° C./36° to 46° F., protected from light, and was not frozen.

Method of Assigning Subjects to Treatment Groups

As they were enrolled in the study, healthy subjects in Part 1 of thestudy were assigned unique consecutive numbers beginning with 1101,1201, 1301 and 1401 for Groups 1, 2, 3, and 4, respectively. Subjectswith mild to moderate controlled asthma in Part 2 were assigned uniqueconsecutive numbers beginning with 2501, 2601 and 2701 for Groups 5, 6and 7, respectively. Subjects with mild to moderate controlled asthma inPart 3 were assigned unique consecutive numbers beginning with 3801 and3901 for Groups 8 and 9, respectively. The subjects were randomlyassigned to receive 13C5.5 or placebo. The randomization schedule wascomputer-generated before the start of the study.

Selection of Doses in the Study

The maximum recommended starting dose (MRSD) for the first-in-human(FIH) trial was calculated according to the US Food and DrugAdministration (FDA) Guidance for Industry “Estimating the safe startingdose in the clinical trials for therapeutics in adult healthyvolunteers.” Per the guidance, MRSD for proteins with molecular weight(MW)>100,000 daltons that are administered IV, should be estimated bynormalizing across species in mg/kg, rather than using body surface areascaling. Additionally, the 13C5.5 in vitro and in vivo data for humanversus cynomolgus monkey rIL-13 or rat rIL-13 demonstrate a ˜8-155-foldpotency shift, respectively which was included in the final MRSDestimates.

Based on the no adverse event effect level (NOAEL) from the 2-weekrepeat dose cynomolgus monkey study, a safety factor of 10, and a 8-foldpotency shift between monkey and human rIL-13 from in vivo pharmacologydata, the MRSD was determined to be 19 mg/kg. Similarly based on theNOAEL from the 4 week repeat dose rat study, a safety factor of 10, anda 52-fold potency shift between rat and human rIL-13 from in vivopharmacology data, the MRSD was determined to be 3 mg/kg. Furthermore,when a potency shift of 155-fold using the in vitro pharmacology data isused, the human MRSD was determined to be 1 mg/kg.

A range of doses were evaluated in this trial to establishdose-linearity, with the low dose selected to allow for an appropriatesafety margin while minimizing the likelihood of immunogenicity. Thehigh dose was chosen to evaluate the safety margin of high doses in botha healthy and controlled asthma population prior to proceeding intoPhase 2 to enable appropriate dose ranging design in theproof-of-concept study. Safety and tolerability were establishedfollowing single escalating doses prior to proceeding to multipledosing. Subcutaneous multiple dosing was important for assessingbioavailability, potential exposure-response relationships in theasthmatic population, and immunogenicity.

Prior and Concomitant Therapy

For Part 1, concomitant medications were not to be permitted throughoutthe study.

For Parts 2 and 3, short acting beta agonist use as rescue therapy waspermitted during the study. Low to medium dose ICS use, and nasalcorticosteroid use was permitted during the study. Long-acting betaagonist use was allowed for patients as prescribed if LABA was beingused concurrently with ICS at Screening. There should have been no planto change asthma medications for the duration of the study.

Systemic corticosteroid use was permitted if needed for control of anasthma exacerbation but subjects were to be discontinued from anyfurther treatment with 13C5.5. Subjects who required use of systemiccorticosteroids were to remain in the study and followed for safetyassessments.

Only essential medications needed for the treatment of existing medicalconditions were allowed during the study. These included lipid-loweringagents, anti-hypertensives, antacids, histamine-2 receptor antagonists,proton pump inhibitors, and pain relievers such as aspirin ornon-steroidal anti-inflammatory drugs (NSAIDs). Antihistamine use,over-the-counter emollient use, and/or low to medium potency topicalcorticosteroid (Class IV, V, VI or VII) use was permitted in subjectswith atopic dermatitis meeting entry criteria. Nasal corticosteroid usewas permitted in subjects with allergic rhinitis. Subjects were onstable doses of permitted medications for a minimum of 8 weeks prior toStudy Day 1 and remained on stable doses throughout the study. Use ofacetaminophen of 2 g or less per day was allowed on an intermittentbasis at the discretion of the investigator.

Use of non-essential medications was discouraged but if a subjectreported taking any over-the-counter or prescription medications,vitamins and/or herbal supplements or if administration of anymedication became necessary from 30 days prior to study drugadministration through the end of the study, the name of the medication,dosage information including dose and frequency, date(s) ofadministration including start and end dates, and reason for use wererecorded, and the medical monitor was notified.

13C5.5 and ADA Assay

Blood samples for 13C5.5 assay and anti-drug antibody (ADA) wereobtained throughout the study, as indicated in Table 3. The time thateach blood sample was collected was recorded to the nearest minute inthe source document and on the appropriate electronic case report form(eCRF). The timing of blood collections took priority over all otherscheduled study activities except for dosing.

Additional blood samples for drug measurement may have been collectedfrom subjects if they were discontinued due to adverse events; the clocktime, and date the sample was taken were to be recorded.

Samples were to be collected within ±5 minutes of the scheduled times onStudy Day 1, within ±1 hour of the scheduled times on Study Days 2through 6, and within ±3 hours of the scheduled times on Study Days 7through 127.

TABLE 3 Serum Volume (mL) for Each Sample IV Doses Hour 3, 4, 6, 10, 14,24, 1344, 48, 72, 96, 2016, 0 2 120, 168 336 504 672 1008 2688 13C5.51.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 (MSD) ADA 1.5 1.5 1.5 1.5 (screeningassay) ADA 2.0 2.0 2.0 2.0 (neutralizing assay) SC Doses Hour 672, 1008,8, 24, 48, 72, 360, 384, 408, 1344, 2016, 0 96, 120, 168, 336 432, 456,504 2352, 3024 13C5.5 1.5 1.5 1.5 1.5 1.5 (MSD) ADA 1.5 1.5 1.5(screening assay) ADA 2.0 2.0 2.0 (neutralizing assay) MSD =Meso-scale-discovery assay.

Results from MSD assay for 13C5.5 and screening assay for ADA werereported for this study.

Blood samples for the 13C5.5 assay were collected by venipuncture intoappropriately labeled evacuated serum collection tubes without gelseparator. For IV study drug administration (Parts 1 and 2), samples for13C5.5 assay were collected at 0 hour (pre-dose) and at 2, 3, 4, 6, 10,14, 24, 48, 72, 96, 120, 168, 336, 504, 672, 1008, 1344, 2016, and 2688hours following the onset of study drug administration on Study Day 1.For SC study drug administration (Part 3), samples for 13C5.5 assay werecollected at 0 hour (pre-dose) and at 8, 24, 48, 72, 96, 120, 168, 336,360, 384, 408, 432, 456, 504, 672, 1008, 1344, 2016, 2352 and 3024 hoursfollowing study drug administration on Study Day 1. The samples forhours 168 and 336 were collected immediately prior to dosing on StudyDays 8 and 15, respectively. A blood sample for 13C5.5 assay wasobtained at the early termination visit, if applicable. The serum volumeneeded for each sample is listed in Table 3. Blood was allowed to clotfor 30 minutes at room temperature before centrifugation.

Blood samples for ADA assay were collected by venipuncture intoappropriately labeled evacuated serum collection tubes without gelseparator. For IV study drug administration, blood samples for ADA assaywere collected on Study Day 1 at 0 hour (pre-dose) and on Study Days 15,29, 57, 85 and 113. For SC study drug administration, blood samples forADA assay were collected on Study Days 1 and 15 at 0 hour (pre-dose) andon Study Days 29, 43, 57, 85, 99 and 127. A blood sample for ADA assaywas also obtained at the early termination visit, if applicable. Theserum volume needed for each sample is listed in Table 3. Blood wasallowed to clot for 30 minutes at room temperature beforecentrifugation.

Pharmacokinetic Variables

Values for the pharmacokinetic parameters of 13C5.5 after IV dosing wereestimated using noncompartmental methods.

The maximum observed serum concentration (C_(max)) and the time toC_(max) (peak time, T_(max)) were determined directly from the serumconcentration-time data.

The value of the apparent terminal phase elimination rate constant (β,Beta) was obtained from the slope of the least squares linear regressionof the logarithms of the serum concentration versus time data from theterminal log-linear phase of the profile. The terminal log-linear phasewas identified using Phoenix™ WinNonlin® Version 6.1 to (PharsightCorporation, Mountain View, Calif.) and visual inspection. A minimum ofthree concentration-time data points was used to determine β. The actualtimes used for each subject may be found in the tables of the calculatedpharmacokinetic parameters. The terminal phase elimination half-life(t_(1/2)) was calculated as ln(2)/β.

The area under the plasma concentration-time curve (AUC) from time 0 tothe time of the last measurable concentration (AUC_(t)) was calculatedby the linear trapezoidal rule. The AUC was extrapolated to infinitetime by dividing the last measurable plasma concentration (C_(t)) by β.Denoting the extrapolated portion of the AUC by AUC_(ext), the AUC fromtime 0 to infinite time (AUC_(∞), AUC_(inf)) was calculated as follows:

AUC _(∞) =AUC _(t) +AUC _(ext)

The percentage of the contribution of the extrapolated AUC (AUC_(ext))to the overall AUC_(∞) was calculated by dividing the AUC_(ext) by theAUC_(∞) and multiplying this quotient by 100.

The clearance value (CL) was calculated by dividing the administereddose by the AUC_(∞). The volume of distribution (Vd_(β), VDB) value wascalculated by dividing the CL by β. An estimate of the volume ofdistribution at steady state (V_(ss)) was also presented.

Dose-normalized C_(max), AUC_(t) and AUC_(∞) were also calculated forall groups in Parts 1 and 2.

For the SC dose groups in Part 3, C_(max), T_(max), and AUC from 0 to168 hours post-dose (AUC₀₋₁₆₈) were estimated after the first and thirddoses. Beta and t_(1/2) were estimated following the third SC dose.Additionally, dose-normalized C_(max) and AUC₀₋₁₆₈ were calculated forGroups 8 and 9 in Part 3. The accumulation ratio (Rac) for the AUC₀₋₁₆₈for Study Day 15 relative to the AUC₀₋₁₆₈ for Study Day 1 was alsocalculated.

Pharmacokinetics

For each of Parts 1, 2 and 3, serum concentrations of 13C5.5 and ADA andpharmacokinetic parameter values were tabulated for each subject andeach dose group, and summary statistics were computed for each samplingtime and each parameter.

For all the 13C5.5 single-dose regimens (groups) in healthy subjects(Part 1), analyses were performed on dose-normalized C_(max),dose-normalized AUC, T_(max) and β to address the issue of linearpharmacokinetics and dose proportionality. The logarithmictransformation was employed for C_(max) and AUC. For each parameter, aone-way analysis of variance (ANOVA) was performed. Subjects wereclassified by dose level. Body weight or another measure of size was tobe included as a covariate in the model for C_(max) and AUC if theregression coefficient was statistically significant at level of 0.10.Within the framework of the final model, the highest dose was comparedto the lowest dose. For the logarithms of C_(max) and AUC, a 95%confidence interval, as well as a point estimate, were provided for theratio of the central value of the highest dose relative to that of thelowest dose. The point estimate and the 95% confidence interval wereobtained by exponentiation of the corresponding estimate and confidencelimits for the difference of logarithm means. If at least four 13C5.5dose levels were studied, a test was also performed at significancelevel 0.05 on a contrast in the dose level means, with the contrastchosen to be sensitive to an approximately linear trend with thelogarithm of dose.

ANOVAs were also conducted for the single dose regimens in subjects withmild to moderate controlled asthma (Part 2). Within the framework of themodel, the hypothesis of no difference between the highest and lowestdoses was tested. The analyses could or could not have been performedjointly (analyses were not performed jointly) for the two populations(healthy subjects and subjects with mild to moderate controlled asthma).If analyses had been performed jointly for the two populations, thenonly data from the dose levels that the two populations had in common,were to be included. In this case, the model would have had effects forpopulation, dose level and the interaction of population by dose level.Body weight or another measure of size could have been included in themodel for C_(max) and AUC. If the statistic on population-doseinteraction had been significant at level of 0.10, then estimates andother inferences would have been provided for each populationseparately. Otherwise, estimates and other inferences were to have beenbased on the dose level main effects.

ANOVA was also performed for the multiple-dose regimens (Part 3) forpharmacokinetic parameters corresponding to those of the single-doseregimens. The decision on whether to include a measure of size as acovariate was based in part on the results for Parts 1 and 2. ForC_(max) and AUC, point estimates and 95% confidence intervals wereprovided for the ratio of the central values of the two doses, asexplained for Part 1.

Missing Values and Model Violations

If there had been missing values due to premature discontinuations thatwere possibly related to study drug, the possibility of bias as a resultof the missing values would have been considered.

Values of pharmacokinetic variables (C_(max), AUC, etc.) are normallydetermined without replacing missing individual concentration values,simply using the available data, and, if necessary, performing theanalysis with some missing values for a pharmacokinetic variable.However, missing concentration values for isolated individual serumsamples could have been replaced (imputed) if such might have affectedstudy conclusions or meaningfully affected point estimates.

If the probability distribution of a variable had non-symmetry to thedegree that conclusions from the ANOVA (ANOVAs were performed for allparts rather than ANCOVAs) might have been affected or point estimateswere misleading, then a transformation (an alternative to the logarithmin the case of C_(max) and AUC) that yielded an approximately symmetricdistribution was to have been sought. If a satisfactory transformationcould not have been found or if it appeared that both tails of theprobability distribution were quite long, a nonparametric analysis couldhave been performed. If the dose levels had unequal variances to theextent that conclusions might have been affected, then approximatemethods that allowed for unequal variances would have been used.

Example 1 Escalating Single Doses in Healthy and Asthma Subjects (Groups1 Through 7)

The mean±standard deviation (SD) serum concentration-time profiles aftera single IV infusion of 0.3 mg/kg to 10 mg/kg 13C5.5 to healthy subjects(Groups 1 through 4) are presented in FIGS. 5 and 6 on linear andlog-linear scales, respectively. The mean±SD serum concentration-timeprofiles after a single IV infusion of 0.3 mg/kg, 3.0 mg/kg or 10 mg/kg13C5.5 to healthy subjects and subjects with mild to moderate asthma(Groups 1, 3 through 7) are presented in FIGS. 7 and 8 on linear andlog-linear scales, respectively.

The mean±SD pharmacokinetic parameters of 13C5.5 after a single IVinfusion of 13C5.5 to healthy and asthma subjects are shown in Table 4.

TABLE 4 Mean ± SD Pharmacokinetic Parameters of 13C

 

 a Single IV Infusion of 13C5.5 in Healthy Subjects and Asthma SubjectsGroup 5 Group 2 Group 3 Group 6 Group 7 Group 1 0.3 mg/kg 1.0 mg/kg 3.0mg/kg 3.0 mg/kg Group 4 10 mg/kg 0.3 mg/kg (N = 4) (N = (N = (N = 4) 10mg/kg (N = 4) (N = 4) Mild/ 3)^(#) 3)^(#) Mild/ (N = 4) Mild/ PK HealthyMod Healthy Healthy Mod Healthy Mod Parameter Unit Volunteer AsthmaVolunteer Volunteer Asthma Volunteer Asthma C_(max) ^(†) μg/mL 6.88 ±0.49 6.72 ± 0.91 20.65 ± 2.43  80.15 ± 6.11  68.55 ± 10.46 214.50 ±27.77  292.00 ± 19.88  AUC_(t) ^(†) μg · hr/mL 2102 ± 495  1999 ± 450 6490 ± 1192 26214 ± 5519  24580 ± 2578  82527 ± 8957  92307 ± 9810 AUG_(∞) ^(†) μg · hr/mL 2264 ± 394  2123 ± 544  6649 ± 1362 27180 ±6051  25037 ± 2913  87342 ± 10966 94917 ± 9276  t_(1/2) ^(¢) day 17.35 ±3.34  17.24 ± 8.24  18.83 ± 4.83  22.64 ± 4.26  16.37 ± 6.35  26.66 ±4.52  23.50 ± 4.45  CL^(†) mL/hr/kg 0.135 ± 0.022 0.148 ± 0.035 0.154 ±0.028 0.114 ± 0.027 0.121 ± 0.013 0.116 ± 0.015 0.106 ± 0.010 Vd_(β)^(†) mL/kg 82.4 ± 12.6 96.6 ± 25.9 102.0 ± 11.9  89.2 ± 7.6  72.8 ± 16.7108.9 ± 18.1  89.0 ± 19.7 V_(ss) ^(†) mL/kg 80.3 ± 4.5  90.0 ± 20.0 95.1± 7.3  83.1 ± 7.0  75.9 ± 15.9 97.7 ± 

69.7 ± 

C_(max)/Dose μg/mL/ 22.93 ± 1.65  22.41 ± 3.02  20.65 ± 2.43  26.72 ±2.04  22.85 ± 3.49  21.45 ± 2.78  29.20 ± 1.99  (mg/kg) AUC_(t)/Dose μg· hr/ 7007 ± 1650 6664 ± 1501 6490 ± 1192 8738 ± 1840 8193 ± 859  8253 ±896  9231 ± 981  mL/(mg/kg) AUC_(∞)/Dose μg · hr/ 7546 ± 1315 7076 ±1812 6649 ± 1362 9060 ± 2017 8346 ± 971  8734 ± 1097 9492 ± 928 mL/(mg/kg) ^(#)N = 4 for C_(max) and C_(max)/Dose for Groups 2 and 3.^(¢)Harmonic mean ± pseudo-standard deviation; evaluations of t_(1/2)were based on statistical tests for β. ^(†)Parameter was not testedstatistically. ^(V) _(ss): Estimate of volume of distribution at steadystate.

indicates data missing or illegible when filed

Following IV administration, the exposures, as determined by AUC andC_(max), appeared to increase in a dose dependent manner over the 0.3mg/kg to 10 mg/kg range. Exposures (AUC and C_(max)) to 13C5.5 inhealthy and asthma subjects were similar at the tested doses (0.3 mg/kg,3.0 mg/kg, and 10.0 mg/kg IV). The pharmacokinetics of 13C5.5 weresimilar to that of a typical immunoglobulin G1 (IgG1) with a smallvolume of distribution and long half-life. Harmonic mean±pseduo SDhalf-lives of 13C5.5 ranged from 16.4±6.35 days to 26.7±4.52 days, andmean Vd_(β) ranged from 72.8 to 108.9 mL/kg following IV infusions overthe 0.3 mg/kg to 10.0 mg/kg dose range.

The total variabilities in C_(max), AUC_(t) and AUC_(∞) for 13C5.5expressed as percent CV for escalating 13C5.5 single IV infusions inhealthy and asthma subjects are shown in Table 5.

TABLE 5 Total Variability for Pharmacokinetic Parameters (Parts 1 and 2)Variability (% CV) Group 1 Group 2 Group 3 Group 4 0.3 mg/kg 1.0 mg/kg3.0 mg/kg 10 mg/kg (N = 4) (N = 3)^(#) (N = 3)^(#) (N = 4) Parameter(Units) Healthy Subjects C_(max) μg/mL 7.2 11.8 7.6 12.9 AUC_(t) μg ·hr/mL 23.5 18.4 21.1 10.9 AUC_(∞) μg · hr/mL 17.4 20.5 22.3 12.6 Mild toModerate Asthma Subjects Group 5 Group 6 Group 7 0.3 mg/kg 3.0 mg/kg 10mg/kg (N = 4) (N = 4) (N = 4) C_(max) μg/mL 13.5 15.3 6.8 AUC_(t) μg ·hr/mL 22.5 10.5 10.6 AUC_(∞) μg · hr/mL 25.6 11.6 9.8 ^(#)N = 4 forC_(max) for Groups 2 and 3.

Example 2 Dose Proportionality and Pharmacokinetic Linearity in Healthyand Asthma Subjects (Parts 1 and 2)

The mean±SD dose-normalized C_(max) and AUC_(∞) values for 13C5.5following administration of single IV infusions of 13C5.5 over the 0.3to 10 mg/kg dose range are presented in Table 4. The mean±SDdose-normalized C_(max) and AUC_(∞) values of 13C5.5 versus dose levelare presented in FIG. 9.

To address the issues of pharmacokinetic linearity and doseproportionality in healthy subjects and subjects with mild to moderateasthma, an ANOVA was performed on the pharmacokinetic parameters.Subjects were classified by treatment group (regimen).

In healthy subjects, 13C5.5 pharmacokinetics were dose linear in C_(max)and AUC. The mean C_(max), AUC_(t) or AUC_(∞) values were similarbetween the highest dose (10 mg/kg) and the lowest dose (0.3 mg/kg).There were no statistically significant trends (p>0.05) for changes in13C5.5 dose-normalized C_(max), or dose-normalized AUC over the doserange (0.3 to 10 mg/kg). However, the power of the tests was low due tosmall number of subjects in each of the dose groups. Based onstatistical tests for β, the t_(1/2) value for the highest dose(26.7±4.52 days, 10 mg/kg) was statistically significantly longer thanthe value of 17.4±3.34 days for the lowest dose of 0.3 mg/kg (Table 4).The results also indicated that there were statistically significanttrends (p<0.05) for increases in 13C5.5 t_(1/2) across the 0.3 to 10mg/kg dose range.

In subjects with mild to moderate asthma, the dose-normalized C_(max) ofthe highest dose (10 mg/kg) was statistically significantly higher thanthat for the lowest dose of 0.3 mg/kg. The dose-normalized AUC_(t) andAUC_(∞) values of the highest dose were statistically significantlygreater (p<0.05) than those of the lowest dose. However, the observeddeparture from dose proportionality was small with an estimate of theratio of central values of 1.3 for C_(max) and 1.4 for AUC over a33-fold range of doses.

Example 3 Three Weekly SC Injections in Asthma Subjects (Groups 8 and 9)

The mean±SD serum concentration versus time profiles of 13C5.5 following3 weekly SC doses of 13C5.5 for Group 8 (0.3 mg/kg) and Group 9 (3.0mg/kg) are presented in FIG. 10 on linear and log-linear scales.

The mean±SD pharmacokinetic parameters of 13C5.5 following three weeklydoses of 0.3 mg/kg or 3.0 mg/kg 13C5.5 SC are shown in Table 6. Theaccumulation following 3 weekly doses of 13C5.5 appears to be linear asthe C_(max) and AUC values are approximately 3-fold greater followingthree 0.3 mg/kg or 3.0 mg/kg weekly doses of 13C5.5. T_(max) appears tobe similar following the first and third 0.3 and 3.0 mg/kg doses of13C5.5. The harmonic mean±pseudo-SD half-life for 13C5.5 was 27.29±3.33and 24.30±1.23 days after SC administration of 0.3 and 3.0 mg/kg forthree days, respectively. Using simultaneous pharmacokinetic modeling ofIV and SC data, the estimated bioavailability of 13C5.5 wasapproximately 70%.

TABLE 6 Mean ± SD Pharmacokinetic Parameters of 13C5.5 Following 3Weekly SC Doses of 13C5.5 in Asthma Subjects Group 8 Group 9 0.3 mg/kg,0.3 mg/kg, 3.0 mg/kg, 3.0 mg/kg, Pharmacokinetic (Dose 1) (Dose 3) Dose3/Dose 1 (Dose 1) (Dose 3) Dose 3/Dose 1 Parameter Unit (N = 4) (N = 4)Rac (N = 4) (N = 4) Rac C_(max) μg/mL 1.80 ± 0.72  4.87 ± 0.97 — 17.90 ±5.91   47.08 ± 12.54 — AUC₀₋₁₆₈ μg · hr/mL 236 ± 106  678 ± 107 3.192278 ± 1083  6875 ± 1343 3.29 T_(max) ^(£) hr 108 (48-168) 84 (49-120) —108 (72-168) 108 (48-169) — t_(1/2) ^(¢) day — 27.29 ± 3.33 — — 24.30 ±1.23 — C_(max)/Dose μg/mL/(mg/kg) 6.01 ± 2.40 16.23 ± 3.23 — 5.97 ± 1.9715.69 ± 4.18 — AUC₀₋₁₆₈/Dose μg · hr/mL/(mg/kg) 788 ± 353 2259 ± 355 —759 ± 361 2292 ± 448 — Rac: Accumulation ratio, calculated as the ratioof Day 15 AUC₀₋₁₆₈ to Day 1 AUC₀₋₁₆₈. ^(£)Median (minimum-maximum).^(¢)Harmonic mean ± pseudo SD.

The total variabilities in C_(max) and AUC₀₋₁₆₈ for 13C5.5 expressfollowing administration of three weekly SC doses of 13C5.5 in subjectswith mild to moderate asthma are shown in Table 7.

TABLE 7 Total Variability for Pharmacokinetic Parameters (Part 3)Variability (% CV), Mild to Moderate Asthma Subjects Group 8 Group 9 0.3mg/kg SC 3.0 mg/kg SC (N = 4) (N = 4) Parameter (Units) Day 1 Day 15 Day1 Day 15 C_(max) μg/mL 39.9 19.9 33.0 26.6 AUC₀₋₁₆₈ μg · hr/mL 44.8 15.747.6 19.5

Example 4 Dose Proportionality and Pharmacokinetic Linearity (Part 3)

The mean±SD dose-normalized C_(max) and AUC∞ values for 13C5.5 followingadministration of 3 weekly SC doses of 13C5.5 0.3 or 3.0 mg/kg arepresented in Table 6. The mean±SD dose-normalized C_(max) and AUC∞values of 13C5.5 versus dose level are presented in FIGS. 11 and 12,respectively.

To address the issues of pharmacokinetic linearity and doseproportionality an ANOVA was performed on the pharmacokineticparameters. Subjects were classified by treatment group (regimen).

In subjects with mild to moderate asthma administered 3 weekly SC doses,13C5.5 pharmacokinetics were dose linear in C_(max) and AUC. The meanT_(max), C_(max) and AUC₀₋₁₆₈ values were similar between the highestdose (3.0 mg/kg) and the lowest dose (0.3 mg/kg) on both Day 1 and Day15. Of note, the power of the tests was low due to small number ofsubjects in each of the dose groups.

Statistics/Analysis for Examples 1-4

Adjustments for covariates were not performed.

Subjects 1202 and 1304 prematurely discontinued from the study and theirlast assay blood samples were collected at Hour 672 and Hour 96,respectively. For these two subjects, pharmacokinetic parameters werenot calculated except for C_(max) and T_(max). Subject 1103 prematurelydiscontinued from the study and his last assay blood sample wascollected at approximately 894 hours post dose. For this subject,pharmacokinetic parameters were calculated. The few cases of a missingconcentration for an individual sampling time did not prevent adetermination of values of the pharmacokinetic parameters in which thereis confidence.

Subjects whose available data warranted determination of values for thepharmacokinetic parameters were included in the statistical analysis.This study was conducted at a single study center; therefore, noconsiderations for a plurality of centers were necessary.

Conclusion for Examples 1-4

The pharmacokinetics of 13C5.5 after single dose administration wereconsistent with that of a IgG1 with a long half-life and small volume ofdistribution. For subjects administered a single IV infusion of 13C5.5,the systemic exposure (AUC and C_(max)) to 13C5.5 increased in a doseproportional manner over the dose range of 0.3 to 10 mg/kg for healthysubjects; however, for subjects with mild to moderate controlled asthma,AUC and C_(max) increased in a slightly more (30 to 40%) than doseproportional manner over the same 33-fold dose range. For subjects withmild to moderate asthma administered 3 weekly SC doses, 13C5.5pharmacokinetics were dose linear in both AUC and C_(max) between the0.3 and 3.0 mg/kg doses. The accumulation of 13C5.5 was as expectedabout 3-fold following three weekly SC doses.

During the study, 13C5.5 was well tolerated and safe when administeredas a single dose up to 10 mg/kg IV or as multiple doses of 0.3 and 3mg/kg SC. The adverse event profile in healthy adults was similar tothat observed in subjects with asthma. No dose-related increases oradministration-specific trends in treatment-emergent adverse events wereappreciated. In each part of the study, the proportion of subjectsreporting an upper respiratory tract infection or viral upperrespiratory tract infection was greater among those receiving 13C5.5compared to those receiving placebo. All of these events were of mild ormoderate severity, and none were judged by the investigator to bepossibly or probably related to study drug. However, the incidence ofrespiratory infections will continue to be monitored closely in futurestudies. Multiple events of blood CPK increased were reported includingin one placebo subject; these events occurred following the initialconfinement at the study site and were associated in each case withtransient elevations in CPK and a history of increased physicalactivity.

One subject in the 13C5.5 0.3 mg/kg treatment group experienced aserious adverse event of hospitalization for bunionectomy that wasassessed by the investigator as not related to study drugadministration. No subjects discontinued study drug due to atreatment-emergent adverse event. Other than a report of infusion sitepain in one subject, there were no adverse events of infusion-relatedreactions reported, and a review of events that could representdeterioration in asthma and spirometry data did not suggest a worseningof underlying disease in asthma subjects.

No clinically significant trends were detected in other safety analysesincluding vital signs, ECG variables and laboratory measurements.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated composition comprising an anti-IL-13 antibody, orantigen-binding portion thereof, wherein, when administeredintravenously to a subject at a dose of about 0.3 mg/kg, the antibody,or antigen-binding portion thereof, is capable of exhibiting: (a) anarea under the curve (AUC) of between about 1,500 and about 2,700μgh/ml; (b) a volume of distribution of between about 65 and 125 mL/kg;(c) a peak concentration (C_(max)) of between about 5 and about 8 μg/ml;and (d) a clearance rate of between about 0.1 and about 0.2 ml/h/kg. 2.An isolated composition comprising an anti-IL-13 antibody, orantigen-binding portion thereof, wherein, when administeredintravenously to a subject at a dose of about 3 mg/kg, the antibody, orantigen-binding portion thereof, is capable of exhibiting: (a) an areaunder the curve (AUC) of between about 21,000 and about 33,500 μgh/ml;(b) a volume of distribution of between about 55 and about 100 mL/kg;(c) a peak concentration (C_(max)) of between about 55 and about 90μg/ml; and (d) a clearance rate of between about 0.08 and about 0.15ml/h/kg.
 3. An isolated composition comprising an anti-IL-13 antibody,or antigen-binding portion thereof, wherein, when administeredintravenously to a subject at a dose of about 10 mg/kg, the antibody, orantigen-binding portion thereof, is capable of exhibiting: (a) an areaunder the curve (AUC) of between about 75 and about 100 μgh/ml; (b) avolume of distribution of between about 90 and about 130 mL/kg; (c) apeak concentration (C_(max)) of between about 185 and about 250 μg/ml;and (d) a clearance rate of between about 0.1 and about 0.15 ml/h/kg. 4.An isolated composition comprising an anti-IL-13 antibody, orantigen-binding portion thereof, wherein, when administeredsubcutaneously to a subject at a dose of about 0.3 mg/kg, the antibody,or antigen-binding portion thereof, is capable of exhibiting: (a) anarea under the curve (AUC) of between about 125 and about 800 μgh/ml;and (b) a peak concentration (C_(max)) of between about 1.0 and about6.0 μg/ml.
 5. An isolated composition comprising an anti-IL-13 antibody,or antigen-binding portion thereof, wherein, when administeredsubcutaneously to a subject at a dose of about 3 mg/kg, the antibody, orantigen-binding portion thereof, is capable of exhibiting: (a) an areaunder the curve (AUC) of between about 1,100 and about 8,500 μgh/ml; and(b) a peak concentration (C_(max)) of between about 12 and about 60μg/ml. 6.-7. (canceled)
 8. A method of treating or preventing asthma ina subject comprising administering the composition of any one of claims1-5 to the subject, thereby treating or preventing asthma in saidsubject. 9.-14. (canceled)
 15. A method of treating asthma in a subjectcomprising intravenously administering to the subject an anti-IL-13antibody, or antigen-binding portion thereof, wherein at least onepharmacokinetic characteristic selected from the group consisting of:(a) a maximum serum concentration (C_(max)) of between about 5 and about235 μg/mL, and (b) an area under the serum concentration-time curve(AUC) of between about 1,500 and about 98,000 μgh/mL, is achievedfollowing administration of the antibody, or antigen-binding portionthereof to said subject.
 16. The method of claim 15, wherein theantibody, or antigen-binding portion thereof, is administered at a doseof about 0.3 mg/kg, optionally wherein the C_(max) is between about 5and about 10 μg/mL, or wherein the AUC is between about 1,500 and about2,700 μgh/mL. 17.-18. (canceled)
 19. The method of claim 15, wherein theantibody, or antigen-binding portion thereof, is administered at a doseof about 3 mg/kg, optionally wherein the C_(max) is between about 55 andabout 90 μg/mL, or wherein the AUC is between about 20,000 and about34,000 μgh/mL. 20.-21. (canceled)
 22. The method of claim 15, whereinthe antibody, or antigen-binding portion thereof, is administered at adose of about 10 mg/kg, optionally wherein the C_(max) is between about190 and about 235 μg/mL, or wherein the AUC is between about 75,000 andabout 100,000 μgh/mL. 23.-26. (canceled)
 27. A method of treating asthmain a subject comprising subcutaneously administering to the subject ananti-IL-13 antibody, or antigen-binding portion thereof, wherein atleast one pharmacokinetic characteristic selected from the groupconsisting of: (a) a maximum serum concentration (C_(max)) of betweenabout 1 and about 60 μg/mL, and (b) an area under the serumconcentration-time curve (AUC) of between about 125 and about 8,100μgh/mL, is achieved following administration of the antibody, orantigen-binding portion thereof to said subject.
 28. The method of claim27, wherein the antibody, or antigen-binding portion thereof, isadministered at a dose of about 0.3 mg/kg, optionally wherein theC_(max) is between about 1 and about 6 μg/mL, or wherein the AUC isbetween about 100 and about 800 μgh/mL. 29.-30. (canceled)
 31. Themethod of claim 27, wherein the antibody, or antigen-binding portionthereof, is administered at a dose of about 3 mg/kg, optionally whereinthe C_(max) is between about 12 and about 60 μg/mL, or wherein the AUCis between about 1,100 and about 8,100 μgh/mL. 32.-40. (canceled)
 41. Amethod of treating asthma in a subject comprising subcutaneouslyadministering to the subject an anti-IL-13 antibody, or antigen-bindingportion thereof, at a dose of about 0.3 mg/kg, wherein at least onepharmacokinetic characteristic selected from the group consisting of:(a) a half-life of between about 24 and 31 days; (b) a T_(max) ofbetween about 3 and about 5 days; and (c) a bioavailability of at leastabout 60%, is achieved following administration of the antibody, orantigen-binding portion thereof to said subject.
 42. (canceled)
 43. Amethod of treating asthma in a subject comprising subcutaneouslyadministering to the subject an anti-IL-13 antibody, or antigen-bindingportion thereof, at a dose of about 3 mg/kg, wherein at least onepharmacokinetic characteristic selected from the group consisting of:(a) a half-life of between about 23 and 26 days; (b) a T_(max) of lessthan or equal to about 5 days; and (c) a bioavailability of at leastabout 60%, is achieved following administration of the antibody, orantigen-binding portion thereof to said subject.
 44. (canceled)
 45. Amethod of treating asthma in a subject comprising intravenouslyadministering to the subject an anti-IL-13 antibody, or antigen-bindingportion thereof, at a dose of about 0.3 mg/kg, wherein at least onepharmacokinetic characteristic selected from the group consisting of:(a) a clearance rate of between about 0.11 to about 0.19 mL/hr/kg; and(b) a volume of distribution of between about 70 to about 130 mL/kg isachieved following administration of the antibody, or antigen-bindingportion thereof to said subject.
 46. A method of treating asthma in asubject comprising intravenously administering to the subject ananti-IL-13 antibody, or antigen-binding portion thereof, at a dose ofabout 3 mg/kg, wherein at least one pharmacokinetic characteristicselected from the group consisting of: (a) a clearance rate of betweenabout 0.08 to about 0.14 mL/hr/kg; and (b) a volume of distribution ofbetween about 55 to about 100 mL/kg is achieved following administrationof the antibody, or antigen-binding portion thereof to said subject. 47.A method of treating asthma in a subject comprising intravenouslyadministering to the subject an anti-IL-13 antibody, or antigen-bindingportion thereof, at a dose of about 10 mg/kg, wherein at least onepharmacokinetic characteristic selected from the group consisting of:(a) a clearance rate of between about 0.09 to about 0.13 mL/hr/kg; and(b) a volume of distribution of between about 85 to about 130 mL/kg isachieved following administration of the antibody, or antigen-bindingportion thereof to said subject.
 48. The composition of claim 1 or themethod of claim 41, wherein the anti-IL-13 antibody, or antigen-bindingportion thereof, is 13C5.5, or an antigen-binding portion thereof. 49.The method of claim 41, wherein the anti-IL-13 antibody, orantigen-binding portion thereof, is administered once.
 50. The method ofclaim 41, wherein the anti-IL-13 antibody, or antigen-binding portionthereof, is administered weekly, optionally wherein the antibody, orantigen-binding portion thereof, is administered for 3 weeks. 51.-52.(canceled)
 53. The method of claim 41, further comprising administeringan additional agent to said subject, optionally wherein said additionalagent is selected from the group consisting of: a therapeutic agent, animaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinaseinhibitor, a co-stimulation molecule blocker, an adhesion moleculeblocker, an anti-cytokine antibody or functional fragment thereof;methotrexate, a cyclosporin, a rapamycin, an FK506, a detectable labelor reporter, a TNF antagonist, an anti-rheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an oralsteroid, an epinephrine or analog, a cytokine, and a cytokineantagonist.
 54. (canceled)
 55. The composition claim 1 or the method ofclaim 15, wherein said subject is a human, or wherein the asthma is mildto moderate asthma. 56.-59. (canceled)